In the final year of this project, we focused on elucidating the mechanisms of leukemia stem cell (LSC) generation in JAK2 compared with BCR-ABL1 initiated myeloproliferative neoplasms (MPN, previously called myeloproliferative disorders). To this end, we investigated the MPN stem cell propagating effects of BCR-ABL1 or JAK2 alone or in combination with activation of the human embryonic stem cell RNA editase, ADAR1. Recently, we discovered that ADAR1, which edits adenosine to inosine bases in the context of primate specific Alu sequences, leads to GSK3β missplicing and β-catenin activation in chronic phase (CP) CML progenitors leading to blast crisis (BC) transformation and LSC generation. In addition, variant isoform expression of a Wnt/β-catenin target gene, CD44, was also characteristic of LSC. In a previous report (Jiang et al., PNAS 2013), identification of ADAR1 as a malignant reprogramming factor represented the first description of RNA editing as a regulator of reprogramming. When lentivirally overexpressed, ADAR1 endows committed CP myeloid progenitors with self-renewal capacity. Further studies revealed that JAK2/STAT5a activates ADAR1 leading to deregulation of cell cycle progression and global down-regulation of microRNA expression thereby uncovering two additional key mechanisms of LSC generation in MPNs. This is consistent with our findings from gene expression profiling studies performed in the previous year, along with functional classification and network analysis using Ingenuity Pathway Analysis (IPA), showing that cell cycle-related genes were significantly altered in human progenitors from xenografted mice treated with combination JAK2 and BCR-ABL inhibitor therapy compared with single agent therapies alone. Together these data suggest that combined BCR-ABL and JAK2 inhibition impairs LSC survival and self-renewal via cell cycle modulation. ADAR1 and other stem cell regulatory pathways such as CD44 represent novel targets to detect and eradicate the self-renewing LSC. We also performed new studies that elucidate the stem cell-intrinsic genetic changes that occur during human bone marrow aging, which may contribute to BCR-ABL or JAK2-dependent functional alterations.
This work has led to discovery of a novel role for embryonic stem cell genes and splice isoforms, including ADAR1 p150 and a transcript variant of CD44, in the maintenance of LSC that promote MPN progression. In addition, through the course of this research we have 1) developed novel lentiviral tools for investigating normal hematopoietic stem and progenitor (HSPC) and malignant LSC survival, differentiation, self-renewal, and cell cycle regulation, and 2) devised innovative LSC diagnostic strategies and 3) tested therapeutic strategies targeting LSC-associated RNA editing and splice isoform generation that selectively inhibit LSC self-renewal.