High-Dimensional Mass Cytometry Analysis Reveals Differential STAT Activation in Cellular Subsets of Chronic Myelogenous Leukemia
Jitakshi De, Neil P Shah, CT Ballentine, Holden T Maecker. Stanford University, Stanford, CA; University of California San Francisco, San Francisco, CA; MTFE Records, Oakland, CA
Background: In chronic myelogenous leukemia (CML), the BCR-ABL translocation results in constitutive STAT5 activation. Despite their susceptibility to tyrosine kinase inhibitors (TKIs), STAT pathways have been implicated in therapy refractoriness. By blocking Bcl6, STAT5 enhances expression of IL6 which has been linked to myeloid expansion and blast crisis, and suppression of lymphoid differentiation. We sought to study cell-specific cytokine-potentiated effects on signaling events in CML by mass cytometry (MC), a powerful new approach to simultaneously quantify cell-specific effects of modulators on multiple oncogenic pathways.
Design: Fresh blood from a untreated 56y male patient with chronic phase CML was obtained from UCSF, and tested in parallel with a healthy donor sample. Aliquots of each were treated with IL3, IL6, IFNα, or no stimulus, for 15 min at 37C followed by surface staining prior to fixation, permeabilization, and intracellular staining. The panel of 27 markers included a series of metal-conjugated antibodies towards lineage-associated markers and phospho-specific epitopes within STAT5, STAT3, p38 MAP kinase, and PLCγ2; and total IκB kinase. Data from MC analysis was analyzed using high-dimensional analytical tools.
Results: Marked potentiated effect of IL3 on p-STAT5 was seen in cells derived from common myeloid progenitors (neutrophils, monocytes, and basophils); but not B- or T-lymphocytes in CML. Compared to normal B-cells, the B-cells in CML had higher baseline p-STAT5, but reduced IL3-STAT5 activity. The IL6-STAT3 pathway demonstrated higher activity in CML monocytes and CD4+ T-cells compared to normal counterparts. A minute IL3R-α expressing CD4+ T-cell subset showed STAT responses similar to myeloid progenitors. IL3-IκB was reduced in putative CD38- CD66+ myeloid blasts, suggestive of increased NFκB activity in this subset.
Conclusions: Findings of the present study offer novel insights into cell-specific oncogenic behavior and open avenues for further studies testing therapeutic role of inhibiting STAT and possibly NFκB in refractory CML. Reduced inducible STAT5 in CML B-cells raises questions regarding targetable STAT5 levels in B-lymphoid blast crisis and Ph+ acute lymphoblastic leukemia. Given the crucial role of IL6 in CML pathogenesis and its elevation secondary to Bcl6 repression, targeting Bcl6 while attractive due to its reactivation in TKI-refractory CML stem cells, must be critically evaluated.
Monday, March 4, 2013 1:00 PM
Poster Session II # 278, Monday Afternoon