Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia

Abstract

Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression. We have interrogated myeloproliferative signaling in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) patient samples using mass cytometry, which allows the quantitative measurement of multiple signaling molecules simultaneously at the single cell level, in cell populations representing a nearly complete spectrum of hematopoiesis. MF and sAML malignant cells demonstrated a high prevalence of hyperactivation of the JAK-STAT, MAP kinase, PI3 kinase, and NFκB signaling pathways. Constitutive NFκB signaling was evident across MF and sAML patients. A supporting GSEA analysis of MF showed many NFκB target genes to be expressed above normal levels in MF patient CD34+ cells. NFκB inhibition suppressed colony formation from MF CD34+ cells. This study indicates that NFκB signaling contributes to human myeloproliferative disease and is abnormally activated in MF and sAML.

Identification of functionally primitive and immunophenotypically distinct subpopulations in secondary acute myeloid leukemia by mass cytometry

“…NFκB signaling has been shown to be a characteristic of undifferentiated cells, further indicating that AMC06 may have some stem/progenitor-like characteristics (36,37). This is consistent with a recent study from our group reporting abnormal activation of NFκB signaling in HSPCs in myelofibrosis and sAML (28).…”

“…S14B). This pathway has recently been demonstrated by our group as being commonly hyperactive in sAML (28). AMC06 also exhibited higher Ki67 levels than normal Lin − CD61 − CD34 + CD38 − cells in all patients examined, suggestive of a higher rate of proliferation (Supporting Information Fig.…”

“…(36,37) This is consistent with a recent study from our group reporting abnormal activation of NFĸB signaling in HSPCs in myelofibrosis and sAML. (28)…”

Integrated functional and mass spectrometry-based flow cytometric phenotyping to describe the immune microenvironment in acute myeloid leukemia

“…Mass cytometry has also been successfully utilized to investigate signaling pathways in AML tumor cells (Han et al, 2015; Fisher et al, 2017; Levine et al, 2015). For example, mass cytometry has been used to associate the functional maturity of AML cells to their surface phenotype and demonstrate that an “immature” signaling profile associates with poor outcome (Levine et al, 2015).…”

“…For example, mass cytometry has been used to associate the functional maturity of AML cells to their surface phenotype and demonstrate that an “immature” signaling profile associates with poor outcome (Levine et al, 2015). An additional example of this type of analysis was recently reported by a group who was able to identify potential signaling pathway targets in patients with myelofibrosis and AML and overlay cell activation status into a hematopoietic map (Fisher et al, 2017). A similar strategy has been used to investigate the signaling impacts of mTOR and bromodomain inhibitors on leukemic stem cell populations in patients with AML (Zeng et al, 2016; Saenz et al, 2017).…”