Poster Session II: Chronic lymphocytic leukemia and related disorders - Biology & translational research
Reversible protein phosphorylation is a dynamic process that regulates virtually all cellular events and is controlled by the opposed activity of protein kinases and protein phosphatases. Although strong emphasis has been placed on the hyperactivation of protein kinases in the onset and progression of human disease, especially cancer, accumulating evidence suggests that abnormal signaling also results from impaired expression or activity of phosphatases. In Large Granular Lymphocyte (LGL) leukemia (LGLL), a rare lymphoproliferative disorder chatracterized by clonal expansion of either cytotoxic T lymphocytes (T-LGLL) or natural killer cells (chronic lymphoproliferative disorder of NK cells, NK-CLPD), constitutive activation of phosphorylation-regulated survival pathways, JAK2/STAT3, PI3K/Akt, and NF-κB pathways among others, has been found to account for the molecular mechanisms sustaining monoclonal cell proliferation and resistance to apoptosis. Interestingly, since several factors participating in such pathways are known to be substrates for protein phosphatases, namely the tyrosine phosphatase SHP-1 and the serine/threonine phosphatase PP2A under normal conditions, it is conceivable that their possible alterations and consequent inability to disrupt hyperactive pathways play a role in LGLL pathogenesis.
In this study, we assessed the ability of small molecules activating SHP-1 and PP2A to abrogate the survival pathways in LGLs by counteracting aberrant signals generated by constitutively activated protein kinases.
Peripheral blood specimens were obtained from untreated patients with LGLL (both T-LGLL and NK-CLPD). Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque (Sigma Aldrich) gradient separation. LGLs were then separated from PBMCs by the immunomagnetic beads (Miltenyi Biotec). LGLs were incubated with SC-43, SHP-1 activator, or alternatively CC11, PP2A activator, at increasing concentrations and different time points. Subsequently, LGLs underwent annexin V-PI flow cytometry to assess the extent of apoptosis or were lysed to monitor the level of SHP-1 and PP2A activity. Moreover, the phosphorylation/activation status and the protein level of factors directly involved in the constitutively activated survival pathways of LGLs were evaluated by Western blot analysis.
Both SC-43 and CC11 proved effective at bringing a significant level of caspase-dependent apoptosis in the low micromolar range, also exhibiting a striking synergistic effect when used in combination. Moreover, both compounds selectively activated their target phosphatases, as demonstrated by proper in-vitro phosphatase assays. As to the signaling pathways affected by SC-43 via SHP-1 activation, the phosphorylation status of STAT3 was remarkably decreased, thereby negatively affecting the function of this transcription factor, as shown by the reduced expression of downstream target genes such as Mcl-1, Cyclin D and S1P5. In turn, CC11 caused dephosphorylation of factors taking part in PI3K/Akt and NF-kB pathways, including Akt itself and IKKbeta and IkB, respectively, confirming the role of PP2A as functional antagonist of survival pathways in patients' LGLs.
Taken together, our results support the newly emerging evidence that the drug-induced activation of SHP-1 and PP2A, already recognized as tumor suppressors in other tumor cells, may serve as a mechanism countering the aberrant pro-survival signals, thus opening up new prospects for treatment of LGLL.