Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/ß-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia.

The Wnt/ß-catenin pathway's significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/ß-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/ß-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active ß-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish's translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/ß-catenin signaling.

Zebrafish Drug Screening Identifies Erlotinib as an Inhibitor of Wnt/ß-Catenin Signaling and Self-Renewal in T-cell Acute Lymphoblastic Leukemia.

The Wnt/ß-catenin pathway's significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential, yet clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex crosstalk with other pathways. In this study, we leveraged the zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/ß-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/ß-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which are known to rely on active ß-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish's translational potential in drug discovery and repurposing, and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/ß-catenin signaling. Highlights: Zebrafish-based drug screening offers an inexpensive and robust platform for identifying compounds with high efficacy and low toxicity in vivo . Erlotinib, an Epidermal Growth Factor Receptor (EGFR) inhibitor, emerged as a potent and promising Wnt inhibitor with effects in both zebrafish and human cell-based Wnt reporter assays.The identification of Erlotinib as a Wnt inhibitor underscores the value of repurposed drugs in developing targeted therapies to disrupt cancer stemness and improve clinical outcomes.

Nanopore sequencing of clonal IGH rearrangements in cell-free DNA as a biomarker for acute lymphoblastic leukemia.

Background: Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer, and patients with relapsed ALL have a poor prognosis. Detection of ALL blasts remaining at the end of treatment, or minimal residual disease (MRD), and spread of ALL into the central nervous system (CNS) have prognostic importance in ALL. Current methods to detect MRD and CNS disease in ALL rely on the presence of ALL blasts in patient samples. Cell-free DNA, or small fragments of DNA released by cancer cells into patient biofluids, has emerged as a robust and sensitive biomarker to assess cancer burden, although cfDNA analysis has not previously been applied to ALL. Methods: We present a simple and rapid workflow based on NanoporeMinION sequencing of PCR amplified B cell-specific rearrangement of the (IGH) locus in cfDNA from B-ALL patient samples. A cohort of 5 pediatric B-ALL patient samples was chosen for the study based on the MRD and CNS disease status. Results: Quantitation of IGH-variable sequences in cfDNA allowed us to detect clonal heterogeneity and track the response of individual B-ALL clones throughout treatment. cfDNA was detected in patient biofluids with clinical diagnoses of MRD and CNS disease, and leukemic clones could be detected even when diagnostic cell-count thresholds for MRD were not met. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient, even when blasts are not physically present in the biofluid sample. Conclusions: The Nanopore IGH detection workflow to monitor cell-free DNA is a simple, rapid, and inexpensive assay that may ultimately serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

Protocol for rapid assessment of the efficacy of novel Wnt inhibitors using zebrafish models.

Dysregulation of Wnt signaling is a hallmark of many cancers, and the development of effective, non-toxic small-molecule Wnt inhibitors is desirable. Off-target toxicities of new compounds are typically tested in mouse models, which is both costly and time consuming. Here, we present a rapid and inexpensive protocol to determine the in vivo toxicity and efficacy of novel Wnt inhibitors in zebrafish using a combination of a fluorescence reporter assay as well as eye rescue and fin regeneration assays. These experiments are completed within 1 week to rapidly narrow drug candidates before moving to more expensive pre-clinical testing. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020).

Epigenetic Regulation of Wnt Signaling by Carboxamide-Substituted Benzhydryl Amines that Function as Histone Demethylase Inhibitors.

Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (ß-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.