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4337 Increased STAT5/STAT3 Intracellular Signaling in Circulating CD34+ Cells of Patients with PMF Correlates with Disease Severity

Program: Oral and Poster Abstracts
Session: 635. Myeloproliferative Syndromes: Basic Science: Poster III
Hematology Disease Topics & Pathways:
Diseases, Adult, Therapies, Biological Processes, MPN, Technology and Procedures, Study Population, Myeloid Malignancies, flow cytometry, signal transduction
Monday, December 3, 2018, 6:00 PM-8:00 PM
Hall GH (San Diego Convention Center)

Carlotta Abbà, MSc1*, Rita Campanelli1*, Paolo Catarsi1*, Laura Villani1*, Vittorio Abbonante, Ph.D.2*, Christian Andrea Di Buduo, Ph.D.2*, Melania Antonietta Sesta3*, Vittorio Rosti, MD1, Giovanni Barosi, MD1 and Margherita Massa3*

1Center for the Study of Myelofibrosis, Laboratory of Biochemistry Biotecnology and Advanced Diagnosis, IRCCS Policlinico San Matteo Foundation, Pavia, Italy
2Department of Molecular Medicine, University of Pavia, Pavia, Italy
3Laboratory of Biochemistry, Biotecnology and Advanced Diagnosis, IRCCS Policlinico San Matteo Foundation, Pavia, Italy

Primary Myelofibrosis (PMF) is a clonal disorder of the hematopoietic progenitor cells (HPCs) characterized by bone marrow (BM) fibrosis, increased number of peripheral blood (PB) CD34+ cells, splenomegaly and increased risk of leukemic transformation. PMF has been associated with driver mutations such as JAK2V617F, Calreticulin (CALR) indels, and MPLW515. All the driver mutations activate the cytokine/receptor JAK2 pathway and its downstream signaling such as the signaling transducer and activator of transcription (STAT1, 3, and 5), phosphatidylinositol 3-kinase (PI3K)/ AKT/mTOR, and the MAPK/ extracellular signal-regulated kinase (ERK) pathways. It has been reported an increased basal signaling of ERK in BM CD34+ HPCs and of STAT5 and STAT3 in BM CD34- cells from patients with PMF; no significant correlation was described between these signaling pathways and mutant JAK2 allele burden (Anand et al Blood 2011;118:1610).

In this study, by a phospho-specific flow cytometry assay, we measured basal and cytokine induced signaling activity of STAT3, STAT5, and ERK1, 2 in the PB CD34+ cells and granulocytes of 53 patients with PMF not receiving therapy: 31 were JAK2V617F mutated (20 heterozygous), 19 CALR+, and 3 MPL+. Thirteen healthy subjects were assessed as controls (CTRLs). STAT3, STAT5, and ERK1, 2 phosphorylation was measured before/following incubation of PB with recombinant IL-6 (Miltenyi Biotec), thrombopoietin (TPO-Peprotech), or phorbol-12 myristate 13- acetate (PMA-Sigma Aldrich) for 15 minutes a 37°C. Red cells were lysed by Lyse/Fix buffer (Becton Dickinson); PB cells were washed and permeabilized by Perm buffer III (BD). The anti human CD34-FITC (BD) was added to all the conditions. Antibodies used for phosphoprotein detection (p-STAT3, p-STAT5, p-ERK1,2) were Alexa Fluor647-conjugated (BD). Isotype controls were used at the same concentrations as test antibodies.

More than 1x106 cells/sample were acquired by a Navios flow cytometer (Beckman Coulter), and analysed by Kaluza software. Both PB CD34+ cells and granulocytes were electronically gated (granulocytes on the basis of physical parameters). Data, calculated as median fluorescence intensity (MFI) test antibody/ MFI isotype control, are shown as (median, range).

We show higher values (p=0.002) of basal p-STAT5 in the PB CD34+ cells of patients (MFI 2.0, 0.99-4.7) than in CTRLs (MFI 1.6, 0.95-2.7), while the STAT3 and ERK1,2 basal phosphorylations are comparable in patients and CTRLs (not shown). Correlations are present between basal p-STAT5 and hemoglobin levels (R= - 0.28, p=0.038), the absolute number of circulating CD34+ cells (R= 0.39, p=0.005), and the percentage of CD34+CXCR4+ cells (R= -0.47, p=0.0007). In addition, basal p-STAT5 of CD34+ cells is significantly correlated (R=0.29, p=0.031) with a severity score including leukocytosis, thrombocytosis, and splenomegaly (myeloproliferation index) and anemia, leukopenia, and thrombocytopenia (myelodepletion index). Basal p-STAT5 is not correlated with mutant JAK2 allele burden. STAT3, STAT5, and ERK1, 2 phosphorylation of granulocytes is comparable in patients and CTRLs (not shown).

TPO induced p-STAT5 (MFI 9.0, 1.0-29) and IL6 induced p-STAT3 values (MFI 7.0, 0.3-14) in CD34+ cells, are higher (p=0.00004 and p=0.0004, respectively) in patients than in CTRLs and are inversely correlated with the percentage of circulating CD34+CXCR4+ cells (R= -0.30, p=0.04 and R= -0.34, p=0.017, respectively). In addition, the induced p-STAT3 values correlate with the severity score (R=0.30, p=0.03) and the absolute number of circulating CD34+ cells (R= 0.34, p=0.014). No correlation is present with mutant JAK2 allele burden. The cytokine induced p-STAT3, p-STAT5, and p-ERK1,2 is comparable in granulocytes from patients and CTRLs.

In conclusion, increased basal STAT5 and induced STAT5/STAT3 phosphorylation characterize circulating CD34+ HPCs, but not granulocytes in patients with PMF. The altered basal STAT5 and induced STAT5/STAT3 phosphorylation is not associated with mutated JAK2 allele burden, but it may affect the disease severity and course. Interestingly, basal STAT5 and induced STAT5/STAT3 phosphorylation appears to modulate CXCR4 expression on CD34+ HPCs. Taken together, our data indicate that the degree of STAT3 and STAT5 activation pathway in PB CD34+ cells of patients with PMF can determine some clinical and biological features of the disease.

Disclosures: No relevant conflicts of interest to declare.

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