Querying Cytokine-Signaling Networks in Myeloproliferative Neoplasms

Mass cytometry helps elucidate the basis of signaling abnormalities in myelofibrosis

Media

Watch this video to hear Dr. Stephen Oh, MD, PhD, of Washington University detail his findings using mass cytometry to investigate altered signaling networks and cellular cytokine expression in subjects with myelofibrosis.

Myeloproliferative neoplasms including myelofibrosis (MF) are characterized by anemia, splenomegaly, bone marrow fibrosis and inflammatory cytokine production. JAK2 inhibition with ruxolitinib improves symptoms and lowers circulating plasma cytokine levels but does not ameliorate anemia, fibrosis or malignant clonal burden. The goal of this study is to better define the relationship between dysregulated cytokines and downstream signaling in MF and to determine how these pathways can be effectively manipulated for therapeutic benefit.

To interrogate altered signaling in MF, we have employed mass cytometry, a novel technology that enables the quantitative analysis of more than 40 parameters at the single-cell level. We identified NF-kB pathway hyperactivation distributed across multiple hematopoietic cell populations, including T cells. Plasma TNFα levels were elevated in these subjects, suggesting that excessive production of TNFα may lead to broad activation of NF-κB in a non-cell-autonomous fashion.

To further elucidate the etiology of systemic NF-κB hyperactivation, we extended our mass cytometry approach to study the cellular distribution of cytokine production in MF. Intracellular levels of several cytokines (including TNFα) were constitutively elevated in MF subjects compared to healthy controls. Supranormal cytokine expression was accentuated by stimulation with PMA/ionomycin or TLR ligands R-848 or Pam3CSK4. PMA/ionomycin increased production of TNFα from MF CD34+ cells and CD33+ immature myeloid cells.

These findings imply that multiple cell populations in MF overexpress inflammatory cytokines and are hypersensitive to potentiating stimuli. Future experiments will identify signaling effects of multiple elevated cytokines, which may underlie MF features that persist despite JAK2 inhibitor therapy.

Jennifer Frahm, PhD, of Fluidigm also presents a brief overview of mass cytometry, the high-parameter, single-cell analysis technology used by Dr. Oh in his research.

Speakers

Stephen Oh

Dr. Stephen Oh, MD, PhD   
Assistant Professor of Medicine
Division of Hematology 
Washington University School of Medicine
St. Louis, MO

Jennifer Frahm

Jennifer Frahm, PhD
Field Applications Scientist
Fluidigm Corporation
South San Francisco, CA

fluidigm

For Research Use Only. Not for use in diagnostic procedures.