-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

931 Global Transcriptome Profiling Identifies a Key Mir-185-PAK6 Axis That Promotes Survival of Leukemic Stem Cells and Drug-Insensitive Blasts in BCR-ABL+ Human Leukemia

Program: Oral and Poster Abstracts
Type: Oral
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: CML Stem Cells And Their Environment
Hematology Disease Topics & Pathways:
Diseases, Biological, Adult, CML, Therapies, Animal models, Biological Processes, Technology and Procedures, cell expansion, Study Population, Xenograft models, Clinically relevant, Myeloid Malignancies, flow cytometry, TKI, stem cells, molecular interactions, RNA sequencing, pathogenesis, signal transduction
Monday, December 3, 2018: 4:30 PM
Room 7B (San Diego Convention Center)

Andrew Wu, BSc1,2*, Lin Hanyang, PhD1,2*, Katharina Rothe, PhD1,3*, Min Chen, MD, PhD1*, Jens Ruschmann, PhD1*, Oleh Petriv, PhD4*, Kieran O'Neill, PhD1*, Tobias Maetzig, MD1*, David Knapp, PhD1,2*, Naoto Nakamichi, MD, PhD5, Ryan Brinkman, PhD1,3*, Inanc Birol, PhD3,6*, Donna L Forrest, MD7*, Carl Hansen, PhD4*, R. Keith Humphries, MD, PhD2,8, Connie J. Eaves, PhD2,3,8 and Xiaoyan Jiang, MD, PhD2,3,9

1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada
2Department of Medicine, University of British Columbia, Vancouver, BC, Canada
3Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
4Center for High-Throughput Biology and Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada
5Terry Fox Laboratory BC Cancer Agency, Vancouver, BC, Canada
6Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada
7Leukemia/Bone Marrow Transplant Program of British Columbia, BC Cancer Agency, Vancouver, BC, Canada
8Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
9Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, BC, Canada

Chronic myeloid leukemia (CML) stem/progenitor cells and BCR-ABL+ acute lymphoblastic leukemia (ALL) blast cells are insensitive to tyrosine kinase inhibitor (TKI) monotherapies. These cells rapidly generate therapy-resistant clones in vitro and in vivo and are often responsible for disease relapse. Therefore, identification of predictive biomarkers and novel treatments that target key molecular events active in leukemic stem cells (LSCs) are needed. MicroRNAs (miRNAs) are small molecules that regulate the gene expression network and are highly deregulated in many cancers. Through global transcriptome profiling, we have recently identified 66 differentially expressed miRNAs in pre-treatment CD34+ stem/progenitor cells from CML patients (n=6) compared to healthy bone marrow (NBM) controls (n=3, adjusted P<0.05); 26 differentially expressed miRNAs were identified between subsequent IM-nonresponders and IM-responders (P<0.05). 21 differentially expressed miRNAs were successfully validated in additional IM-responders (n=11), IM-nonresponders (n=11) and NBM (n=11). Interestingly, miR-185 was discovered to be one of the most highly deregulated miRNAs, with significant reduction in CD34+ cells from IM-nonresponders compared to IM-responders (p=0.0006). This significant change was further demonstrated in CD34+ cells from CML patients (n=60) before and after 3-month TKI nilotinib treatment in a clinical trial (p<0.05). We further demonstrated that miR-185 functions as a tumor suppressor; its restored expression by lentiviral transduction in CD34+ IM-nonresponder cells significantly impaired survival of these cells and sensitized them to TKI treatment in vitro. Restored miR-185 expression in BCR-ABL+ ALL blasts led to a profound decrease in leukemia burden and significantly enhanced survival compared to controls in vivo (median survival 65 vs. 47 days, P=0.0005). Strikingly, mice injected with miR-185-transduced cells and treated with dasatinib (DA) survived much longer than recipients of control cells treated with DA (median survival 83 vs. 60 days, P=0.0018). Moreover, restoration of miR-185 expression combined with DA treatment greatly reduced in vivo long-term regenerative activity of LSCs from IM-nonresponders as compared to control cells treated with DA in NRG mice (<0.2% vs. 5% GFP+ patient cells in the BM, 25 weeks post-transplantation). We observed not only a marked reduction in GFP+CD34+ cells, but also a near elimination of GFP+CD34+CD38- LSCs that were transduced with miR-185 and treated with DA compared to control cells treated with DA, indicating that restored miR-185 expression combined with DA preferentially prevents the growth of patient-derived long-term leukemia-initiating cells in vivo. Several miRNA target genes were further identified by integrating miRNA expression profiles with gene expression profiles from the same patient samples using strand-specific RNA-seq. Based on three out of six prediction algorithms (mirBase, TargetScan, miRanda, tarBase, mirTarget2, and PicTar), PAK6, a serine/threonine-protein kinase, was found to be highly expressed in CD34+ IM-nonresponder cells compared to IM-responders (p<0.003), which correlated with reduced expression of miR-185 in these cells (p=0.0002). PAK6 was confirmed as a target gene of miR-185 by a luciferase reporter assay. Western blot analysis showed that restored miR-185 expression caused a marked decrease in protein levels of PAK6 in miR-185-transduced cells and suppression of PAK6 reduced viability of these cells. These results indicate that PAK6 is a critical target of miR-185, and that loss of miR-185 expression in CML may lead to up-regulation of PAK6, which in turn contributes to disease progression and drug resistance. Indeed, the use of a pre-clinically validated pan PAK inhibitor (PF-3758309) significantly inhibited the growth of IM-resistant cells and CD34+ IM-nonresponder cells and these effects could be enhanced by TKIs (p<0.05). Mechanistically, we observed that p-ERK and p-AKT were significantly reduced in PAK6 knockdown or miR185-restored IM-resistant cells in response to IM treatment. Thus, we infer that downregulation of PAK6 may sensitize TKI-resistant cells to TKI therapy through inhibition of the RAS/MAPK pathway. Taken together, PAK6, a novel target of miR-185, emerges as an attractive druggable target for combination therapy of TKI-resistant patients.

Disclosures: Wu: Novartis: Research Funding.

Previous Abstract | Next Abstract >>
*signifies non-member of ASH