Cytokine-Mediated Signaling Is Suppressed in Myeloid Cells and Enhanced in Lymphatic Cells in Patients with Chronic Myeloid Leukemia (CML) — Partial Normalization with Tyrosine Kinase Inhibitors.
Authors: Jalkanen Sari , Mustjoki Satu , Porkka Kimmo , Vakkila Jukka 
Published: October 2019

Abstract
Abstract 2180
Poster Board II-157

Introduction.
Aberrant phosphorylation of the BCR-ABL1 tyrosine kinase (TK) is characteristic of chronic myeloid leukemia (CML). This oncoprotein interacts directly with intracellular signaling proteins, alters the responsiveness of cytokine receptors and regulates secretion of autocrine cytokines. Targeted inhibition of BCR-ABL1 with TK inhibitor (TKI) imatinib mesylate (IM) is the current standard treatment of CML. For overcoming IM resistance or intolerance, 2nd generation TKIs (nilotinib, dasatinib) with broader kinase inhibition profile have been approved for clinical use. Although in vitro results suggest that TKIs are immunosuppressive, no increases in opportunistic infections or secondary malignancies have been observed to date. In contrast, in some TKI-treated patients immunoactivation in the form of chronic lymphocytosis linked to excellent therapy responses has recently been shown. Dynamic monitoring of aberrant cytokine signaling pathways would aid in understanding and predicting the development of TKI-resistance or adverse/off-target effects. The aim of this study was to analyze the responsiveness of leukocytes to cytokine stimuli in CML patients at diagnosis and during TKI therapy using single-cell profiling of phosphoprotein networks by multiparameter flow cytometry. Patients and methods. The study consisted of 4 healthy controls, 6 CML patients at diagnosis, 6 IM patients and 5 dasatinib patients. Stimuli included GM-CSF, IL-2+IL-10+IFNα and IL-4+IL-6+IFNγ and they were added immeadately to freshly drawn whole blood ex vivo. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD4+ CD25neg T helper cells (Th), CD4neg lymphocytes and CD4+CD25+ T cells including regulatory T-cells (Treg). Analysis was performed with heatmap function of Cytobank software (http://cytobank.stanford.edu/public/).

Results.
Unstimulated phosphoprotein levels reflecting the activation state of leukocytes in vivo did not differ between healthy controls and CML patients at diagnosis or during dasatinib therapy. Strikingly, in IM patients, baseline levels of pSTAT3 were relatively high indicating in vivo occurring activation of leukocytes in this patient group. We next studied ex vivo responsiveness of immune effector cells with cytokines and found clear differences between healthy controls and CML patients.

At CML diagnosis.
GM-CSF/pERK1+pSTAT5a, IFNa/pSTAT1,and IL-4/pSTAT6 (stimulus/readout) as well as pSTAT3 responses with all stimuli were suppressed in monocytes. In granulocytes, GM-CSF/pSTAT1 levels were diminished. In Th and Treg lymphocytes, IL-6/pSTAT3 responses were markedly pronounced, while IL-10/pSTAT3 responses were not affected when compared to healthy controls. Such difference was not observed in CD4neg lymphocytes.

During TKI therapy.
Most patients (9/11) were in cytogenetic remission at the time of analysis. The unresponsiveness of myeloid cells at diagnosis was restored by IM or dasatinib therapy in most, but not all patients. Similarly, in Th and Treg lymphocytes TKI-therapy normalized the enhanced IL-6/pSTAT3 responses that were evident at diagnosis. However, in Th and Treg cells pSTAT3 responses provoked by IL-10 were particularly prominent. Interestingly, one dasatinib patient with aberrant constant blood NK-lymphocytosis and monocytosis had uniquely strong IFNg/pSTAT1 and IL-4/pSTAT6 responses in monocytes. Furthermore, one patient who have stayed in persistent remission after IM discontinuation had exceptionally high pSTAT3 responses with all of stimuli used. Similar kind of signaling profile was unseen with the other patients and could reflect immunoactivation related to leukemia control.

Conclusions.
Dynamic single-cell profiling of signaling networks is feasible in CML patients and can be used to study mechanisms of aberrant immune reactivity in TKI-treated patients. The method could be particularly suitable for assessing candidate patients for TKI discontinuation. Although in vitro results suggest immunosuppressive effects of TKIs on lymphocytes, leukocytes ex vivo from patients were able to respond similarly to cytokine stimuli as in healthy controls.

Disclosures:
Mustjoki: BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.