Characterization of potential autophagy-related therapeutic targets for FLT3-ITD+ AML with zebrafish models

Abstract

Acute myeloid leukemia (AML) is a severe type of acute leukemia with a higher risk of death and relapse. Many gene mutations and altered signaling pathways have been identified which account for the mechanisms underlying the malignant proliferation of myeloid progenitor cells. Among the mutations, internal tandem duplication (ITD) found in FMS-like tyrosine kinase 3 (FLT3) is reported to be associated with elevated autophagic activity, signaling activity, and cell proliferation in AMLs that show poor prognosis. Autophagy is an important and preserved lysosomal degradation pathway responsible for breaking down cellular components to maintain cellular structure and homeostasis, thus promoting growth and development. Targeting autophagy in AML is a promising therapeutic strategy, while conventional inhibitors of autophagy induce stress on normal hematopoiesis and healthy cells. To develop more effective chemotherapy in AML treatments, novel targets in autophagy or selective autophagy pathways are worth exploring. Zebrafish (Danio rerio) is an advantageous high-throughput vertebrate model for hematopoiesis studies. With primary and definitive hematopoiesis clearly mapped, the location and migration of each lineage can be easily tracked in zebrafish. Many transgenic zebrafish models of human leukemia have been created for pre-clinical studies. Besides hematopoiesis, zebrafish is also widely used to study autophagy. With well-established assays and transgenic reporter lines, zebrafish is an ideal model to study autophagy in AML in vivo. In this project, potential autophagy-related targets were first identified in AML cell lines after treatment with autophagy modulators. Through mass spectrometry-mediated proteomic profiling, I found that the protein levels of the dedicator of cytokinesis (DOCK2), endoplasmic Reticulum Metallopeptidase 1 (ERMP1), and retinoid­inducible serine carboxypeptidase (SCPEP1) were substantially altered by autophagy modulations in the FLT3-ITD+ AML cells. Furthermore, I also discovered another regulator, PTEN-induced putative kinase 1 (PINK1), which was shown to mediate mitophagy/autophagy in hematopoietic cells. These newly discovered genes are potential autophagy-related therapeutic target candidates for FLT3-ITD+ AML. Next, the roles of these genes in AML proliferation and normal hematopoiesis were further investigated in AML cell lines and zebrafish embryos, respectively, through CRISPR/cas9-mediated gene knockout. My results demonstrate that DOCK2 deficiency disrupted autophagy and impaired proliferation of FLT3-ITD+ AML cells, while eliciting negligible effects on FLT3-WT AML cells. Loss of dock2 in zebrafish had little effect on leukocyte development, while remarkably inhibiting the development of neutrophils and lymphocytes. ERMP1 deficiency robustly inhibited autophagy and proliferation of AML cells. Zebrafish ermp1 mutants showed suppressed definitive hematopoiesis (leukocyte and neutrophil development). SCPEP1 deficiency also inhibited leukemia cell proliferation while desensitizing AML cells to the chloroquine treatment. Loss of scpep1 in zebrafish mainly suppressed the development of neutrophils and lymphocytes. On the contrary, PINK1 deficiency enhanced autophagy and cell proliferation of leukemia cells. pink1 loss-of-function mutation promoted the aberrant development of hematopoietic cells through modulating autophagy in zebrafish. In conclusion, I have provided compelling experimental evidence demonstrating the remarkable roles of a group of novel autophagy-associated genes in regulating leukemia cell proliferation and zebrafish hematopoiesis. DOCK2, ERMP1, and SCPEP1 can potentially be targeted for the treatment of AML, while the functional role of PINK1 warrants further examination. My thesis work, which has generated extrapolatable data, has laid a solid foundation for the development of novel therapeutic strategies in AML. The findings of this study certainly have transitional significance and will form a basis for developing further clinical investigations by exploiting autophagy inhibitors for treating AML or other types of cancers.