Proliferation and survival signaling from both Jak2-V617F and Lyn involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 cell line newly established from acute myeloid leukemia transformed from polycythemia vera

Abstract

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),-16,+21,-22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.

Figure 1. Morphological, cytogenetic, and genetic analyses of primary leukemic and PVTL-1 cells.

A) A cytospin preparation of PVTL-1 cells. May-Grunwald-Giemsa staining. B) A bone marrow smear sample at leukemic transformation. C) Giemsa-banded karyotype showing 44,XX,del(5)(q?).-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22.+mar1. Arrows indicate structurally abnormal chromosomes. D, E) Direct sequence analysis of the JAK2 gene obtained by PCR from the peripheral blood of the patient at leukemic transformation (D) and from PVTL-1 cells (E). Nucleotide sequences around the codon coding for V617 in normal Jak2 or F617 in the Jak2 mutant are shown with the mutated codon indicated.

Figure 2. JakI-1 or dasatinib reduces proliferation and viability of PVTL-1 cells.

A, B) PVTL-1 cells were plated at 5×10⁵ cells/ml in RPMI1640 medium with 10% FCS and 1 µM JakI-1, 100 nM dasatinib, or 5 µM imatinib, as indicated, for indicated days. Viable cell numbers and viability were counted and plotted in A and B, respectively. C, D) PVTL-1 or HEL cells, as indicated, were cultured with indicated concentrations of JakI-1 (C) or dasatinib (D) for 3 days and viable cell numbers were measured by XTT colorimetric assay. Each data point represents the mean of triplicate determinations, with error bars indicating standard errors, and is expressed as a percentage of cell numbers without inhibitors. E) PVTL-1 cells were cultured with indicated concentrations of JakI-1, dasatinib, or both, as indicated, for 3 days and analyzed by XTT colorimetric assay. Combination index (CI) values obtained by the method of Chou and Talalay are indicated.

Figure 3. JakI-1 or dasatinib induces apoptosis involving loss of mitochondrial membrane potential and activation of caspase-3 in PVTL-1 cells.

PVTL-1 cells were untreated as control (Cont.) or treated with 1 µM JakI-1 or 10 nM dasatinib, as indicated, for 48 h. Cells were then analyzed by flow cytometry for mitochondrial membrane potential with DiOC6, activation of caspase-3 with the cleavage-specific antibody, surface expression of Annexin V with anti-Annexin V, and cellular DNA content with PI, as indicated. Percentages of cells affected are indicated.

Figure 4. Tyrosine phosphorylation of cellular substrates and formation of signaling complexes by Jak2-V617F and Lyn in PVTL-1 cells.

A–D) PVTL-1 cells were left untreated as control or treated with 1 µM JakI-1, 5 µM imatinib, or 100 nM dasatinib, as indicated for 6 h and lysed. Total cell lysates (A) and immunoprecipitates obtained with anti-Lyn (B), anti-Jak2 (C), or anti-CrkL (D) were subjected to Western blot analyses with antibodies against indicated proteins. Abbreviations used are: PY, phosphotyrosine; STAT5-PY, phospho-Y694-STAT5; Lyn-PY, phospho-Y396-Lyn; Jak2-PY, phospho-Y1007/1008-Jak2; TCL, total cell lysate; IP, immunoprecipitation. An asterisk indicates the position of the most significantly tyrosine-phosphorylated protein. Positions of tyrosine-phosphorylated and non-phosphorylated CrkL, CrkL-PY and CrkL-non-PY, respectively, are indicated. Positions of molecular weight markers are also indicated. E, F) PVTL-1 cells were left untreated as control or treated with 1 µM JakI-1or 100 nM dasatinib, as indicated for 6 h. Cell lysates were subjected to immunoprecipitation with anti-SHP1, anti-SHP2 (E), and anti-Gab2 (F) followed by Western blot analyses. Positions of relevant proteins are indicated.

Figure 5. Jak2-V617F- and Lyn-mediated signaling events in PVTL-1 and HEL cells.

A–E) PVTL-1 or HEL cells were treated with 1.5 µM JakI-1 or dasatinib at 20 nM (A, C, E) or 15 nM (B, D), as indicated, for 6 h (A, C, E) or 1 h (B, D) and lysed. Total cell lysates were subjected to Western blot analyses using indicated antibodies. Abbreviations used are: Jak2-PY, phospho-Y1007/1008-Jak2; STAT5-PY, phospho-Y694-STAT5; Lyn-PY, phospho-Y396-Lyn; 4EBP1-nonP, non-phospho-T46-4EBP1; 4EBP1-P, phospho-T37/46-4EBP1; Erk-P, phospho-T202/Y204-Erk; mTOR-P, phospho-S2448-mTOR; S6K-P, phospho-T389-p70S6K; S6RP-P, phospho-S240/244-S6RP; eIF4B-P, phospho-S422-eIF4B, GSK3-P, phospho-S21/9-GSK3α/ß. F) PVTL-1 cells were precultured with or without 5 µM SB216763 for 1 h and further cultured with or without 1.5 µM JakI-1 or 20 nM dasatinib, as indicated, for 24 h. Cells were analyzed for cellular DNA content with PI by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. G) PVTL-1 cells were precultured with or without 10 µM SB216763 for 1 h and further cultured with or without 1.5 µM JakI-1 or 20 nM dasatinib, as indicated, for 24 h. Cells were lysed and subjected to Western blot analyses using indicated antibodies. Cl. Casp-3: Cleaved Caspase-3.

Figure 6. A hypothetical model of intracellular signaling mechanisms downstream of Jak2-V617F and Lyn regulating survival and proliferation of PVTL-1 cells.

Arrows and dotted lines indicate stimulatory and inhibitory effects, respectively.