The main objectives of this project are generation of myeloproliferative disorder or neoplasm (MPN) stem cells from pluripotent human embryonic stem cells (hESC) or multipotent stem cells, and identification of crucial leukemia stem cell (LSC) survival and self-renewal factors that contribute to the development and progression of BCR-ABL and JAK2-driven hematopoietic disorders. A key finding of our work thus far is that in addition to activation of BCR-ABL or JAK2 oncogenes, generation of self-renewing MPN LSC requires stimulation of other pro-survival and self-renewal factors such as β-catenin, Sonic hedgehog (SHH), BCL2, and in particular the RNA editing enzyme ADAR1, which we identified as a novel regulator of LSC differentiation and self-renewal.
We have now completed comprehensive gene expression analyses from next-generation RNA-sequencing studies performed on normal and leukemic human hematopoietic progenitor cells from primary cord blood samples and adult normal peripheral blood samples, along with normal cord blood transduced with BCR-ABL or JAK2 oncogenes, and primary samples from patients with BCR-ABL+ chronic phase and blast crisis chronic myeloid leukemia (CML). These studies revealed that gene expression patterns in survival and self-renewal pathways (SHH, JAK2, ADAR1) clearly distinguish normal and leukemic progenitor cells as well as MPN disease stages. These data provide a vast resource for identification of LSC-specific biomarkers with diagnostic and prognostic clinical applications, as well as providing new potential therapeutic targets to prevent disease progression.
New results from RNA-sequencing studies reveal high levels of expression of inflammatory mediators in human blast crisis CML progenitors and in BCR-ABL transduced normal cord blood stem cells. Moreover, expression of the inflammation-responsive form of ADAR1 correlated with generation of an abnormally spliced GSK3β gene product that has been previously linked to LSC self-renewal. These results have now been published in the journal PNAS (Jiang et al.). Together, we have demonstrated that ADAR1 drives hematopoietic cell fate by skewing cell differentiation – a trend which occurs during normal bone marrow aging – and promotes LSC self-renewal through alternative splicing of critical survival and self-renewal factors. Notably, inhibition of ADAR1 through genetic knockdown strategies reduced self-renewal capacity of CML LSC, and may have important applications in treatment of other disorders that transform to acute leukemia. Thus, these results suggest that RNA editing (ADAR1) and splicing represent key therapeutic targets for preventing LSC self-renewal – a primary driver of leukemic progression.
Whole transcriptome profiling studies coupled with qRT-PCR, hematopoietic progenitor assays and in vivo studies have shown that combined inhibition of BCR-ABL and JAK2 is another effective method to reduce LSC self-renewal in pre-clinical models. New results show that lentivirus-enforced BCR-ABL or JAK2 expression in normal cord blood stem cells drives generation of distinct splice isoforms of STAT5a. While inhibition of JAK2/STAT5a signaling or BCR-ABL tyrosine kinase activity alone did not eradicate self-renewing LSC, combined JAK2 and BCR-ABL inhibition dramatically impaired LSC survival and self-renewal in the protective bone marrow niche, and increased the lifespan of serial transplant recipients. These effects were associated with reduction in STAT5a isoform expression – which represents a novel molecular marker of response to combined BCR-ABL/JAK2 inhibition – and altered expression of cell cycle genes in human progenitor cells harvested from the bone marrow of transplanted mice. These results are the subject of a new manuscript currently under review (Court et al.). Moreover, this work has led to the development of new experimental tools that will facilitate study of LSC maintenance and cell cycle status in the context of normal versus diseased bone marrow microenvironments. In sum, studies completed thus far have uncovered a role for RNA editing and splicing alterations in leukemic progression, particularly in specific microenvironments. Using specific inhibitors targeting BCR-ABL and JAK2, along with strategies to block RNA editing and aberrant splicing activities, we have been able to establish the relative susceptibility of MPN stem cells to molecular inhibitors with activity against LSC residing in select hematopoietic niches that are difficult to treat with conventional chemotherapeutic agents.