Therapeutic Targeting of Leukemia Stem Cells to Prevent T-Cell Acute Lymphoblastic Leukemia Relapse

The survival rate of T-cell Acute Lymphoblastic Leukemia (T-ALL) relapse is a dismal 10% of affected adults and 30% of children, largely due to the relapsed disease being more aggressive and treatment resistant than the initial disease. Relapse is thought to occur because conventional chemotherapies are unable to reliably eliminate a unique cell type known as leukemia stem (or propagating) cells (LSCs). LSCs are the only cells within the leukemia with the ability to self-renew and remake or replenish the ALL from a single cell. Currently, the pathways governing self-renewal in LSCs are largely unknown, precluding our ability to successfully and selectively target this important cell type with anti-cancer drugs. More research is needed to identify targetable pathways and develop new technologies for studying LSCs. Here, I determined that the oncogenic phosphatase of regenerating liver 3 (PRL-3) plays a role in leukemia progression, migration, and self-renewal of LSCs in T-ALL in vivo in a zebrafish Myc-induced T-ALL model, while inhibition of PRL-3 reduced LSC numbers in vivo and in vitro. RNA sequencing and GSEA of patient T-ALL samples revealed that PRL-3’s role in self-renewal is at least partly due to activation of Wnt pathway signaling, a known driver of LSC function in T-ALL. While the Wnt pathway seems an ideal target for LSCs, Wnt signaling is critical for many normal and developmental processes. Clinical trials for Wnt inhibitors have shown undesirable toxicity and these drugs are not practical for use in children with T-ALL due to developmental concerns. Thus, a major gap in knowledge concerning leukemia stem cells in T-ALL is the identification of regulators of Wnt signaling, like PRL-3, that are uniquely expressed by leukemia cells and easily targeted with small molecules. To expand my research beyond PRL-3, I have developed a novel zebrafish T-ALL model where Wnt expressing cells fluorescently labeled. These animals can be used as a model for studying LSC function and identifying novel drugs that can target Wnt-expressing T-ALL cells in vivo. I have also developed novel translational technologies that may be used to predict LSC driven relapse in T-ALL. I have optimized a zebrafish larval xenograft model for transplant and rapid drug screening of human T-ALL cell lines and patient samples to gain insight into tumor progression and resistance to chemotherapy. I have also developed a novel pipeline for using cell-free circulating tumor DNA (ctDNA) as a biomarker of disease relapse in patients with ALL, enabling tracking of disease course, assessment of minimal residual disease, and as a potential predictor of patient relapse. Taken together, my research has established PRL-3 as a potential therapeutic target in T-ALL, and provided new insight into the role of a PRL-3/Wnt signaling axis in regulating LSC self-renewal. Additionally, the new models and techniques that I have developed are useful tools in analyzing LSC function, targeting self-renewal, and predicting ALL relapse

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)

Protein Tyrosine Phosphatase 4A3 (PTP4A3/PRL-3) Drives Migration and Progression of T-Cell Acute Lymphoblastic Leukemia in Vitro and in Vivo

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer. There are no immunotherapies and few molecularly targeted therapeutics available for treatment of this malignancy. The identification and characterization of genes and pathways that drive T-ALL progression are critical for the development of new therapies for T-ALL. Here, we determined that the protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) plays a critical role in T-ALL initiation and progression by promoting leukemia cell migration. PRL-3 is highly expressed in patient T-ALL samples at both the mRNA and protein levels compared to normal lymphocytes. Knock-down of PRL-3 expression using short-hairpin RNA (shRNA) in human T-ALL cell lines significantly impeded T-ALL cell migration capacity in vitro and reduced their ability to engraft and proliferate in vivo in xenograft mouse models. Additionally, PRL-3 overexpression in a Myc-induced zebrafish T-ALL model significantly accelerated disease onset and shortened the time needed for cells to enter blood circulation. Reverse-phase protein array (RPPA) and gene set enrichment analysis (GSEA) revealed that the SRC signaling pathway is affected by PRL-3. Immunoblot analyses validated that manipulation of PRL-3 expression in T-ALL cells affected the SRC signaling pathway, which is directly involved in cell migration, although Src was not a direct substrate of PRL-3. More importantly, T-ALL cell growth and migration were inhibited by small molecule inhibition of PRL-3, suggesting that PRL-3 has potential as a therapeutic target in T-ALL. Taken together, our study identifies PRL-3 as an oncogenic driver in T-ALL both in vitro and in vivo and provides a strong rationale for targeted therapies that interfere with PRL-3 function

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

BackgroundAcute 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.MethodsWe 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.ResultsQuantitation 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.ConclusionsThe 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

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 (β-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\u27s 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