Abstract
Abstract 1216
Chronic myeloid leukemia (CML) begins as an indolent chronic-phase (CP) when a hematopoietic stem cell (HSC) expresses BCR-ABL, an oncogenic tyrosine kinase generated by the translocation t(9;22)(q34;q11) (i.e. Philadelphia chromosome, Ph). At this phase, BCR-ABL confers a proliferative/survival advantage to CD34+/CD38- primitive HSCs without affecting their multi-lineage capacity, as CD34+/CD38+ committed myeloid precursors (CMPs) are expanded but all BCR-ABL+ blood cells differentiate and function normally. Moreover, tyrosine kinase inhibitors of BCR-ABL (i.e., imatinib mesylate or dasatinib) induce durable remission in CML CP patients but do not eradicate leukemia-initiating cells, which therefore represent a reservoir of disease and potential source of relapse if therapy is stopped or discontinued. These findings strengthen the importance of unravelling HSC mechanisms that control early events on BCR-ABL+ leukemogenesis and therapy-responsiveness of primitive leukemic progenitors, being likely dictated not only by the BCR-ABL itself, but also by the specific HSC microenvironment that the oncogene targets. Here, we focus on glycogen synthase kinase-3β (GSK-3β), a nutrient-responsive kinase that is mainly regulated by serine (inhibitory) and tyrosine (stimulatory) phosphorylation in normal HSCs. It is known that GSK-3β plays multifaceted roles in haematopoiesis, by suppressing WNT/β-catenin pathway responsible for HSC maintenance on the one hand, or by promoting HSC survival and self-renewal through NF-kB activation on the other. Emerging evidence indicates GSK-3β targeting as an effective therapeutic approach in MLL leukemia, Alzheimer's disease and other neurodegenerative diseases, and administration of GSK3β inhibitors in vivo as a clinical means to augment the repopulating capacity of normal HSCs. We here describe balanced levels of transient inactive/active forms of GSK-3β phosphorylated at both serine9 (Ser9) and tyrosine216 (Y216) in normal CD34+ cells versus constitutive levels of active GSK-3β phospho-Y216 in total CD34+ cells and more primitive CD34+CD38- progenitors (<5% of total CD34+ cells) freshly-isolated from CML CP patients. Low or undetectable levels of GSK-3β phospho-Ser9 in primary BCR-ABL+ progenitors indicated that the kinase could be no longer regulated physiologically (e.g. defective inactivation). Imatinib 1mM, corresponding to plasma levels achieved in treated CML CP patients, effectively inhibited BCR-ABL kinase activity but enhanced GSK-3β phospho-Y216 without affecting total GSK-3β expression. Interestingly, increased GSK-3β phospho-Y216 correlated with up-regulated levels of active MAPK kinases in imatinib-treated CML progenitors, as part of a compensatory response induced by exogenous growth factor (GF)-stimulation for maintaining HSC viability. Indeed, imatinib did not change and, however, failed to increase GSK-3β phospho-Y216 in GF-starved CML CP progenitors. Immunofluorescence microscopy was then performed to test changes on the subcellular distribution of GSK-3β in response to BCR-ABL or cytokine stimulation. In normal CD34+ cells depleted of exogenous growth factors, GSK-3β was located predominantly in the cytoplasm, and 2h exposure to GF promoted its nuclear localization. In contrast, CML CD34+ cells showed a cytosolic retention of GSK-3β in presence of GF. By using imatinib (as a selective ABL inhibitor) and dasatinib (as a dual SRC/ABL inhibitor), we showed that CML progenitors can finely integrate oncogenic (BCR-ABL-dependent) and physiological survival signals (via GF-receptor engagement of SRC kinases), to control GSK-3β phospho-Y216, its subcellular distribution and signalling impact on downstream regulators of HSC maintenance (C/EBPα and p27), HSC viability (pp60SRC) and autophagy induction (mTOR). More importantly, our data point at therapeutic targeting of GSK-3β activity by SB-216763 combined with imatinib to strongly increase apoptosis in primary CML CD34+ cells resistant to the elimination by imatinib alone, while sparing normal HSCs. To sum up, this work gains new insights in the biology of primary BCR/ABL+ progenitors and earmarks GSK-3β as an attractive therapeutic target to overcome imatinib resistance in CML CP patients.
Disclosures:
No relevant conflicts of interest to declare.
GSK-3 Is Activated by the Tyrosine Kinase Pyk2 during LPA1-mediated Neurite Retraction
