Terry Fox Research Institute

ABSTRACT The Cancer Dependency map project (DepMap) has created an unprecedented resource of siRNA and CRISPR genetic screens across more than 1000 cell line models. Numerous computational tools have been developed to analyze DepMap outputs and have revealed new genetic dependencies relevant to cancer. Here we extracted correlation information from the DepMap using curated gene sets of biological pathways to determine if this approach could reveal new high confidence candidates. Beginning with a published set of curated DNA repair proteins we determined correlations of query gene knockout fitness across all DepMap cell lines. As expected, this approach extracted many known genome stability genes and also suggested several additional candidates not previously linked to genome maintenance. To validate our analysis we used siRNA depletion for candidate genes and identified several factors whose depletion increases DNA damage or delay repair. Among these was WDR89. WDR89 relocalizes to the nucleolus in an ATM dependent manner upon DNA damage and physically interacts with a number of nucleolar proteins. Depletion of WDR89 also abrogates nuclear p53 accumulation upon irradiation, suggesting a role in p53 signaling. These data establish WDR89 as a new genome stability regulator and highlight the value of DepMap for predicting previously unknown biology.

IntroductionIncidental findings (IF) can be yielded in genomic research that analyzes data from healthy participants or patients. The need for clear IF policies grows along with the increase in international genomic research, data collection and sharing efforts. This study aims to inform policy discussions and decisions about IF.MethodsWe interviewed key stakeholders involved in Canadian, European, or international research projects. We used the 3-I framework for interview design and analysis, which distinguishes interests (i.e., agendas), ideas (i.e., values), and institutions (i.e., policy structures, e.g., laws) as factors that impact policy decisions. We integrated the three bioethical principles into this framework: respect for persons, beneficence, and justice.ResultsInterviewees were from Canada (n = 7) and Europe (n = 4). Different IF policies are followed and practiced. Policy decisions are impacted by varying interests, ideas, and institutions. Prioritization of distinct interests and ideas varies between policies. Key policy considerations are: determining whose interest is prioritized; determining what is of best interest to the participant; determining who is responsible for what when research and healthcare institutional frameworks create tension; determining what is just and a fair distribution of benefits and burdens between individuals and populations; and determining how scarce time and money should be allocated.DiscussionExplicating policy considerations can help to further discuss and decide how IF policies will impact not only research participants, but also patients, citizens, professionals, the public and diverse populations. Technologies, cultural values, and laws and regulations evolve over time. Therefore, continuous discussions should be held.

As DNA image cytometry and DNA image histology serve as valuable tools in clinical tumor pathology, the need for precise and accurate DNA amount measurements is crucial. This study describes the process of employing spectral unmixing on Thionin-stained slides as a means of reducing refraction effects introduced in the image, during imaging, due to changes in the refraction index within the tissue being imaged. A correction method that reduces refraction effects on the DNA quantitation measurements by making use of the spectrally limited absorption properties exhibited by Thionin relative to the more spectrally uniform effects of tissue refraction as a function of wavelength. Spectral unmixing enables an improved estimate of DNA amount at every pixel and a potentially truer representation of the actual distribution of the DNA within individual cell nuclei. Spectral unmixing is a valuable computational technique widely used in histology and cytology research. By reducing refraction-based optical artifacts in the image, it enhances the accuracy of DNA quantitation, minimizes variability, and improves the discriminating ability of nuclear DNA organization as quantified by texture features.

BackgroundT cell acute lymphoblastic leukemia is a fatal hematological malignancy. Despite the treatment progress, no targeted therapy is available currently, which urges to deepen the understanding of the underlying mechanism of T-ALL cell growth/survival. Autophagy is a conserved cellular process, which plays a dual role in human cancers. Nevertheless, many aspects of the involvement of autophagy in T-ALL are not fully understood.MethodsT-ALL patient cells and normal control cells were subjected to RT‒qPCR analysis. Gene silence and overexpression was used to study the function of ATG4B and sestrin 3 (SESN3) in T-ALL cells. Atg4b deficient mice were used to study the role of Atg4b in normal hematopoietic cells and T cell development. The efficacy of S130, an ATG4B inhibitor to suppress T-ALL cell growth was evaluated in xenograft models.ResultsThe results showed that the expression of several autophagy-related genes (especially ATG4B) was significantly higher in T-ALL patient cells than control cells. ATG4B ablation decreased autophagic flux and inhibited T-ALL cell growth. In contrast, Atg4b depletion had mild effects on normal hematopoiesis and T cell development. RNA-seq data and subsequent studies revealed a novel regulatory circuit of ATG4B and SESN3, and the results indicated that SESN3 hampered T-ALL cell growth via the inhibition of both mTOR/S6K/protein synthesis pathway and autophagy. Importantly, S130 exhibited anti-leukemia activity in xenograft models.ConclusionsThe present study demonstrates that a novel ATG4B-SESN3 regulatory circuit plays a crucial role in T cell leukemogenesis, which suggests that targeting ATG4B is a promising strategy for T-ALL treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03588-0.

Tissue-resident memory T (TRM) cells stably occupy tissues and contribute to immunosurveillance, where they are associated with favorable survival outcomes in solid tumors. Although TRM cells have been observed in lymph nodes, their phenotype and prognostic significance in diffuse large B-cell lymphoma (DLBCL) remains uncharacterized. In this study, CD103+ T cells were quantified in DLBCL samples by immunofluorescence staining of tissue biopsies and flow cytometry of cell disaggregates and linked with clinical outcomes. Across two patient cohorts, CD103+ T cells were identified in both nodal and extranodal DLBCL, and higher CD103+ T-cell levels correlated with superior clinical outcomes. Single-cell RNA sequencing revealed ITGAE-expressing T cells in both malignant and reactive lymph node samples. However, transcriptional profiles differed as a canonical TRM population was exclusively observed in the malignant setting. This TRM cluster was enriched for genes associated with cytotoxicity and activation and was validated in an external cellular indexing of transcriptomes and epitopes by sequencing (CITEseq) dataset. Flow cytometry additionally confirmed protein expression of TRM markers (CXCR6, CD39, and PD-1) on CD69+CD103+ T cells. We assessed functional activity in coculture experiments of CD103+ versus CD103- T cells with autologous CD20+ B cells, in which CD103+ T cells displayed enhanced killing. CD103+ TRM cells in DLBCL represent a prognostically favorable population with an activated/cytotoxic T-cell phenotype.

Nuclear medicine imaging allows for a wide variety of data acquisition and image generation methods in the clinical setting. Imaging phantoms are routinely used to evaluate and optimize image quality and quantitative accuracy of features, but few phantoms realistically model the anatomy or heterogeneity of target regions within patient images, such as tumours that are commonly observed in oncology. We developed a negative cast modelling (NCM) technique which enables applications such as non-standard shape tumour phantoms, organ phantoms for radiation dosimetry, and quality control phantoms with small lesions. Tumour templates were derived from segmented PET images of primary mediastinal B-cell lymphoma (PMBCL) patients. Lesion segmentations were saved and 3D-printed. Negatives were developed using silicone-based molding materials, and final models cast using a composition of liquid plastic, pigment, and PET radiotracer. Images of lesions were acquired using the GE DMI PET/CT scanner, and image features were quantified. Mean absolute error (MAE) for tumour volume between the original template and casted models is 13.8%, indicating that the method is reasonably accurate. The high viscosity of the liquid plastic used in the casting process establishes non-uniform tumour models, which is very useful in practice for evaluating image features related to heterogeneity. PET images using the NCM method is determined to be highly realistic by an experienced nuclear medicine physician, due to the non-standard shapes that can be established within the tumours. The NCM method has potential to enable more realistic phantom studies within nuclear medicine imaging. The cost for the lymphoma tumour phantom study is less than $400 USD, making it feasible for large-scale studies.

Effective siRNA delivery in acute lymphoblastic leukemia (ALL) is limited by preferential hepatic accumulation. To address this, a lipopolymer (PEI‐C) is developed by conjugating lipid to polyethylenimine and formulated lipopolymer nanoparticles (LPNPs) via complexation with siRNA. The siRNA delivery efficiency of LPNPs is evaluated in vitro in t(4;11)‐positive ALL cells (RS4;11 and SEM) as well as “normal” peripheral blood mononuclear cells (PBMCs) from human donors and bone marrow stromal cells (BMSCs) from mice. Molecular effects are assessed by quantifying target mRNA silencing and downstream apoptosis. In vivo biodistribution and therapeutic efficacy are examined in mouse models. LPNPs demonstrated significantly higher siRNA uptake than commercial reagents in PBMCs and BMSCs, reaching siRNA uptakes of 87.2% and 93.0% in RS4;11 and SEM cells, respectively. Molecular analyses revealed effective silencing of KMT2A::AFF1 mRNA (≈80% in RS4;11), accompanied by BCL2 downregulation and increased apoptosis. In vivo, LPNPs showed efficient siRNA biodistribution to leukemia‐associated organs (spleen and bone marrow) and significantly reduced leukemia burden in a systemic RS4;11 xenograft mouse model and improved survival. These findings suggest that PEI‐C‐formulated LPNPs present a promising avenue for therapeutic siRNA delivery in ALL, effectively targeting leukemia‐associated organs, and warrant further exploration in clinical studies.

While DNA:RNA hybrids contribute to multiple genomic transactions, their unscheduled formation is a recognized source of DNA lesions. Here, through a suite of systematic screens, we rather observed that a wide range of yeast mutant situations primarily triggering DNA damage actually leads to hybrid accumulation. Focusing on Okazaki fragment processing, we establish that genic hybrids can actually form as a consequence of replication-born discontinuities such as unprocessed flaps or unligated Okazaki fragments. Strikingly, such “post-lesion” DNA:RNA hybrids neither detectably contribute to genetic instability, nor disturb gene expression, as opposed to “pre-lesion” hybrids formed upon defective mRNA biogenesis, e.g., in THO complex mutants. Post-lesion hybrids similarly arise in distinct genomic instability situations, triggered by pharmacological or genetic manipulation of DNA-dependent processes, both in yeast and human cells. Altogether, our data establish that the accumulation of transcription-born DNA:RNA hybrids can occur as a consequence of various types of natural or pathological DNA lesions, yet do not necessarily aggravate their genotoxicity.

Background/Objectives: Overexpressed in various solid tumors, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake of the current clinically evaluated GRPR-targeted radiopharmaceuticals limits their applications. In this study, we replaced the Pro14 residue in our previously reported GRPR-targeted LW02056 and ProBOMB5 with 4,4-difluoroproline (diF-Pro) to obtain an agonist LW02060 (DOTA-Pip-[D-Phe6,Tle10,NMe-His12,diF-Pro14]Bombesin(6-14)) and an antagonist LW02080 (DOTA-Pip-[D-Phe6,NMe-Gly11,Leu13(ψ)diF-Pro14]Bombesin(6-14)), respectively. Methods/Results: The binding affinities (Ki) of Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 were measured by in vitro competition binding assays using PC-3 cells and were found to be 5.57 ± 2.47, 21.7 ± 6.69, 8.00 ± 2.61, and 32.1 ± 8.14 nM, respectively. The 68Ga- and 177Lu-labeled ligands were obtained in 36-75% decay-corrected radiochemical yields with >95% radiochemical purity. PET imaging, SPECT imaging, and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice. Both [68Ga]Ga-LW02060 and [68Ga]Ga-LW02080 enabled clear tumor visualization in PET images at 1 h post-injection (pi). Tumor uptake values of [68Ga]Ga-LW02060 and [68Ga]Ga-LW02080 at 1 h pi were 16.8 ± 2.70 and 7.36 ± 1.33 %ID/g, respectively, while their pancreas uptake values were 3.12 ± 0.89 and 0.38 ± 0.04 %ID/g, respectively. Compared to [177Lu]Lu-LW02080, [177Lu]Lu-LW02060 showed higher tumor uptake at all time points (1, 4, 24, 72, and 120 h pi). However, fast tumor clearance was observed for both [177Lu]Lu-LW02060 and [177Lu]Lu-LW02080. Conclusions: Our data demonstrate that [68Ga]Ga-LW02060 is promising for clinical translation for the detection of GRPR-expressing tumor lesions. However, further optimizations are needed for [177Lu]Lu-LW02060 and [177Lu]Lu-LW02080 to prolong tumor retention for therapeutic applications.