Poor cytokine-induced phosphorylation in chronic myeloid leukemia patients at diagnosis is effectively reversed by tyrosine kinase inhibitor therapy

doi:10.1016/j.exphem.2010.09.005

Experimental Hematology

Volume 39, Issue 1, January 2011, Pages 102-113.e1

https://doi.org/10.1016/j.exphem.2010.09.005 Get rights and content

Objective

In chronic myeloid leukemia (CML), uncontrolled tyrosine kinase activity of the BCR-ABL1 oncoprotein results in aberrant signaling pathways and increased cell proliferation. Acquired immune tolerance to leukemic antigens further enables tumor cell expansion. Tyrosine kinase inhibitor (TKI) therapy interferes with the immunoregulatory system by targeting off-target kinases both in malignant and nonmalignant cells. The aim of this study was to analyze the immune cell function by phosphoprotein profiling in CML patients.

Materials and Methods

Blood samples from diagnostic phase and TKI-treated patients were analyzed by multicolor phosphoprotein flow cytometry enabling measurements at the single-cell level. Both unstimulated baseline activation status and cytokine-induced responses were evaluated.

Results

In diagnostic-phase and imatinib-treated patients, the baseline phosphoprotein activation status was similar to healthy controls. In dasatinib-treated patients, basal phosphoprotein levels were slightly decreased; in particular, the signal transduction and activator of transcription protein 3 pathway was affected in both myeloid and lymphoid cells. The activation responses to various cytokines, granulocyte-macrophage colony-stimulating factor in particular were significantly suppressed in untreated CML patients. During imatinib and dasatinib therapy, the aberrantly suppressed phosphorylation responses were normalized.

Conclusions

Cytokine responses are hampered in untreated CML patients, which may have an effect on various immunological processes in vivo. Interestingly, during TKI treatment, phosphorylation responses were normal, suggesting that TKI treatment does not alter the reactivity of healthy immune effector cells. However, dasatinib treatment was associated with diminished basal activation of the immunosuppressive signal transduction and activator of transcription protein 3 signaling pathway, which could have clinical significance in reversing the lymphocyte anergy against tumor cells.

Section snippets

Patients

The study was comprised of 7 healthy controls with normal white blood cell counts, 10 CML patients at diagnosis, 10 imatinib- and 10 dasatinib-treated CML patients. Four of the diagnostic-phase patients were included in the analysis under imatinib treatment also (patients 8, 9, 12, and 15) and two patients under dasatinib treatment (patient nos. 14 and 17) (Table 1). All patients were in chronic phase. One additional diagnostic-phase patient was excluded from the analysis because none of the

Feasibility of the single-cell phosphoprotein analysis in patient samples

All analyses were performed in conditions mimicking as closely as possible the in vivo signaling environment in patients. Whole blood was used to avoid analytical artifacts due to, for example, delays in cell fractioning. After the venipuncture, blood was immediately placed at +37°C and cytokine stimulations were performed within 1 hour ex vivo. Different steps in sample preparation and analysis (permeabilization of the cell membrane, titration of the antibodies, and cytokines) were optimized

Discussion

Phosphospecific flow cytometry or phosphoflow provides a high-content cell-based platform for immunological monitoring in heterogeneous primary cell populations at the single-cell level 24, 30, 31. The method enables simultaneous identification of various cell types in blood and analysis of their activation state. We previously tested the applicability of the assay for immunological monitoring by analyzing pSTAT1 status in peripheral blood monocytes in hepatitis patients on IFN-α regimen [28].

Acknowledgments

The authors would like to thank Minna Pajuportti (Hematology Research Unit, Helsinki, Finland) for sample preparation and Maija Peltoperä and Taina Huttunen (Huslab, Laboratory of Hematology, Helsinki University Central Hospital, Helsinki, Finland) for expert technical assistance with the flow cytometry. This work was supported by the Finnish special governmental subsidy for health sciences, research and training, by the Finnish Cancer Societies, Emil Aaltonen Foundation, Academy of Finland,

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Moreover, the roles of other factors, such as IL-10 or tumor necrosis factor-α in plasma and other STATs (e.g., pSTAT5 or pSTAT6) and granzyme B in NK cells remain to be elucidated. The response of lymphocytes to cytokines is impaired at the diagnostic stage of CML but is restored by TKI therapy [12]. With regard to the significance of the factor-mediated Th1 response for NK cell activation in dasatinib-treated patients, a rapid decline in tumor burden is presumed to induce an immune response that can lead to minimal residual disease eradication.
We investigated the effects of dasatinib on natural killer (NK) cell-induced signaling protein and perforin expression as well as plasma cytokine levels by analyzing blood samples from patients with well-controlled chronic myeloid leukemia receiving tyrosine kinase inhibitor (TKI) therapy. Perforin expression and phosphorylation of signal transducer and activator of transcription (STAT) 1, STAT3, Janus kinase (JAK) 1, and JAK2 in NK cells were evaluated by flow cytometry, and the levels of plasma cytokines, including interferon (IFN)-γ and interleukin (IL)-2, were determined by enzyme-linked immunosorbent assays in 40 patients (dasatinib, n = 23; imatinib, n = 11; and nilotinib, n = 6). Perforin levels in NK cells were higher in dasatinib-treated patients before TKI treatment; phospho (p)-STAT1 levels were closely correlated with pJAK1 and perforin levels, and pSTAT3 levels were correlated with pJAK2 and perforin levels. The correlation between pJAK1 and pSTAT1 was apparent in dasatinib-treated patients but not in other TKI-treated patients, and the correlation between pJAK2 and pSTAT3 was apparent in patients treated with other TKIs. Constitutive expression of IFN-γ was higher in patients treated with dasatinib or with other TKIs than in those who were in treatment-free remission (TFR). In contrast, constitutive expression of IL-2 was lower in patients treated with other TKIs than in those treated with dasatinib or in those who were in TFR. These results provided insights into the effects of dasatinib on JAK/STAT signaling in NK cells in vivo and the mechanisms underlying NK cell activation induced by dasatinib therapy.
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Signaling by the BCR-ABL fusion kinase drives Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myelogenous leukemia (CML). Despite their clinical activity in many patients with CML, the BCR-ABL kinase inhibitors (BCR-ABL-KIs) imatinib, dasatinib, and nilotinib provide only transient leukemia reduction in patients with Ph+ ALL. While host-derived growth factors in the leukemia microenvironment have been invoked to explain this drug resistance, their relative contribution remains uncertain. Using genetically defined murine Ph+ ALL cells, we identified interleukin 7 (IL-7) as the dominant host factor that attenuates response to BCR-ABL-KIs. To identify potential combination drugs that could overcome this IL-7–dependent BCR-ABL-KI–resistant phenotype, we screened a small-molecule library including Food and Drug Administration–approved drugs. Among the validated hits, the well-tolerated antimalarial drug dihydroartemisinin (DHA) displayed potent activity in vitro and modest in vivo monotherapy activity against engineered murine BCR-ABL-KI–resistant Ph+ ALL. Strikingly, cotreatment with DHA and dasatinib in vivo strongly reduced primary leukemia burden and improved long-term survival in a murine model that faithfully captures the BCR-ABL-KI–resistant phenotype of human Ph+ ALL. This cotreatment protocol durably cured 90% of treated animals, suggesting that this cell-based screening approach efficiently identified drugs that could be rapidly moved to human clinical testing.
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In diagnostic-phase CML patients, the phosphorylation levels of neither STAT5 isoforms were significantly elevated when compared to healthy controls. This is in accordance with our previous publication, showing that newly diagnosed CML patients have normal phosphorylation levels of STAT5a in blood leukocytes when analyzed by phosphoprotein flow cytometry [29]. However, when suboptimal and optimal patients were compared separately with the healthy controls in this study, the suboptimal patients had significantly increased STAT5b Y699 phosphoprotein levels, whereas the optimal patients had similar levels as healthy controls (Supplementary Figure E6; online only, available at www.exphem.org).
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View full text

ImmunobiologyPoor cytokine-induced phosphorylation in chronic myeloid leukemia patients at diagnosis is effectively reversed by tyrosine kinase inhibitor therapy

Objective

Materials and Methods

Results

Conclusions

Section snippets

Patients

Feasibility of the single-cell phosphoprotein analysis in patient samples

Discussion

Acknowledgments

Lancet

J Biol Chem

Blood

Cell

Cytokine

Blood

Blood

Blood

Cancer Cell

Cell

J Immunol Methods

Leuk Res

Clin Immunol

Blood

Blood

Cytokine

Blood

Exp Hematol

Clin Immunol

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis

Leukemia

Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl

J Exp Med

Aberrant cytokine signaling in leukemia

Oncogene

Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia

Nat Med

Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells

J Clin Invest

Phenotypic and functional deficiencies of leukaemic dendritic cells from patients with chronic myeloid leukaemia

Br J Haematol

Target spectrum of the BCR-ABL inhibitors imatinib, nilotinib and dasatinib

Leuk Lymphoma

Immunobiology
Poor cytokine-induced phosphorylation in chronic myeloid leukemia patients at diagnosis is effectively reversed by tyrosine kinase inhibitor therapy