Margarete Zaborski

FLT3 mutations in acute myeloid leukemia cell lines

Internal tandem duplications (ITD) and D835 point mutations of the receptor tyrosine kinase (RTK) FLT3 are found in a high proportion of cases with acute myeloid leukemia (AML). These genetic aberrations may lead to the constitutive activation of the receptor, thus providing the molecular basis for a persisting growth stimulus. We have screened 69 AML-derived cell lines for FLT3 mutations. Four of these cell lines showed ITD of the FLT3 gene, none carried a D835 point mutation. Two cell lines (MUTZ-11 and MV4-11) expressed exclusively the mutated allele, the other two cell lines (MOLM-13 and PL-21) displayed a mutated and the wild-type version of the gene. Although mutationally activated FLT3 is supposed to substitute for the stimulatory signal of a growth factor, one of these cell lines (MUTZ-11) was strictly cytokine-dependent. FLT3 transcripts were found in all four cell lines, but the constitutively phosphorylated receptor protein was clearly detectable only in cell line MV4-11, possibly explaining why MUTZ-11 cells were growth-factor dependent. Thus, not all FLT3 ITD-positive cells express high levels of the active receptor protein, a finding that might be of relevance for a possible future application of a kinase inhibitor as therapeutic agent. It had been described that STAT-5 phosphorylation was part of the FLT3 signalling chain and that STAT-5 molecules were constitutively phosphorylated in FLT3 ITD-positive cells. Although we observed the constitutive phosphorylation of STAT-5 molecules in FLT3-mutant cells, FLT3 ligand (FL) did not induce STAT-5 phosphorylation in FLT3 wild-type cells. These results suggest that the signalling mechanisms of the mutated FL receptor differ at least to some extent from those conferred by wild-type FLT3. In conclusion, (1) not all cells with FLT3 ITD express significant amounts of the mutated receptor protein; (2) signals downstream from wild-type and mutant FLT3 receptors are not 100% idential; and (3) MV4-11 represents a model cell line for FLT3 ITD signalling.

Cloning of human thymic stromal lymphopoietin (TSLP) and signaling mechanisms leading to proliferation.

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that was found to promote the development of murine B cells in vitro. Here we describe the cloning and characterization of the human homologue of murine TSLP. This protein, which is expressed in a number of tissues including heart, liver and prostate, prevented apoptosis and stimulated growth of the human acute myeloid leukemia (AML)-derived cell line MUTZ-3. Anti-interleukin (IL)-7 receptor antibodies (Abs) neutralized this effect indicating that TSLP binds to at least part of the IL-7 receptor complex. TSLP induced phosphorylation of signal transducer and activator of transcription (STAT)-5. In contrast to IL-7, TSLP-triggered STAT-5 phosphorylation was not preceded by activation of janus kinase (JAK) 3. These findings would be in accordance with the notion, raised previously for the mouse system, that TSLP leads to STAT-5 phosphorylation by activating other kinases than the JAKs. Some other signaling pathways stimulated by many cytokines are not involved in TSLP activity; thus, TSLP did not stimulate activation of ERK1,2 and p70S6K. Furthermore, neutralizing Abs raised against cytokines known to stimulate the growth of MUTZ-3 cells did not inhibit the proliferative effects of TSLP, suggesting that TSLP-induced growth was a direct effect. In summary, we describe the cloning of human TSLP and its proliferative effects on a myeloid cell line. TSLP-induced proliferation is preceded by phosphorylation of STAT-5, but not of JAK 3.

JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders.

A mutation in the JH2 pseudokinase domain of the Janus kinase 2 gene (JAK2 V617F) has been described in chronic myeloproliferative disorders (MPD). We screened 79 acute myeloid leukemia (AML) cell lines and found five positive for JAK2 V617F (HEL, MB-02, MUTZ-8, SET-2, UKE-1), 4/5 with histories of MPD/MDS. While SET-2 expressed both mutant (mu) and wild-type (wt) JAK2, remaining positives carried homo-/hemizygous JAK2 mutations. Microsatellite analysis confirmed losses of heterozygosity (LOH) affecting the JAK2 region on chromosome 9p in MB-02, MUTZ-8 and UKE-1, but also in HEL, the only JAK2mu cell line lacking any reported MPD/MDS history. All five JAK2mu cell lines displayed cytogenetic hallmarks of MDS, namely losses of 5q or 7q, remarkably in 4/5 cases affecting both chromosomes. Our combined FISH and microsatellite analysis uncovered a novel mechanism to supplement mitotic recombination previously proposed to explain JAK2 LOH, namely chromosome deletion with/without selective JAK2mu amplification. Confirming the importance of the mutated JAK2 protein for growth and prevention of apoptosis, JAK2mu cell lines displayed higher sensitivities to JAK2 inhibition than JAK2wt cell lines. In summary, JAK2 V617F cell lines, derived from patients with history of MPD/MDS, represent novel research tools for elucidating the pathobiology of this JAK2 mutation.

The human bladder carcinoma cell line 5637 constitutively secretes functional cytokines

We have studied cytokine secretion by the human bladder carcinoma cell line 5637 using growth factor-dependent cell line bioassays and specific ELISA. The 5637 cell line-conditioned medium (5637 CM) stimulated proliferation of human growth factor-dependent leukemia cell lines in a dose-dependent fashion. Quantitation by specific ELISA and by bioassays detected high amounts of G-CSF and GM-CSF and smaller quantities of IL-1beta, M-CSF and SCF in 5637 CM; the concentration of IL-3 was below the detection level of the ELISA, if present at all. The G-CSF and GM-CSF activities secreted by 5637 cells could be inhibited specifically by neutralizing anti-G-CSF and anti-GM-CSF antibodies. In conclusion, 5637 bladder carcinoma cells constitutively produce and secrete several functionally active cytokines; 5637 CM is a valuable, reliable and inexpensive source for cytokines, for instance for the culture of growth factor-responsive or -dependent hematopoietic primary cells and cell lines.

BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance

BackgroundThe BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, de novo or secondary TKI-resistance is a significant problem in CML. We screened a panel of BCR-ABL1 positive ALL and CML cell lines to find models for imatinib-resistance.ResultsFive of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are BCR-ABL1 downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than BCR-ABL1 might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.ConclusionWe introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of BCR-ABL1 or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in PIK3CA, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.

Cytokine response profiles of human myeloid factor-dependent leukemia cell lines

Research in cytokine biology has grown exponentially in recent years as cytokines (often also termed growth factors) are now known to be involved in a wide range of pathological and physiological processes. Continuous human leukemia cell lines represent powerful tools to investigate these mechanisms. Most cell lines grow autonomously in standard culture media (containing fetal bovine serum) independent of externally added growth stimuli. Over the last 5–10 years a battery of myeloid leukemia-derived cell lines has been established that is constitutively dependent on the addition of cytokines to the culture. Such factor-dependent cell lines die rapidly by apoptosis when deprived of the appropriate growth factor. We determined the cytokine response profiles of 19 absolutely growth factor-dependent leukemia cell lines with myelomonocytic, erythroid or megakaryocytic phenotypes with regard to enhanced or suppressed cellular proliferation. Cells were incubated in liquid culture with optimal concentrations of various recombinant human cytokines known to have effects on the growth of hematopoietic cells. A proliferative or anti-proliferative response to these 41 cytokines was assessed by the short-term 3H-thymidine uptake assay. A proliferative response was considered as positive when the stimulation index (SI) was >2; inhibition was regarded as significant with an SI <0.5. the response profile of each cell line to these 41 cytokines was different and individual. none of the cell lines responded to one or two factors only (minimum to at least five cytokines). proliferation of most (n = 13–17), but not of all cell lines was significantly enhanced by GM-CSF, IL-3, PIXY-321, SCF and IFN-γ. TGF-β 1 consistently inhibited proliferation (in 11/19 cell lines). IFN-α, IFN-β, TNF-α and TNF-β had either stimulatory or inhibitory effects. The cell lines responding most often proliferatively (to 15–19 different cytokines) were UCSD/AML1, HU-3, TF-1 and M-07e. In summary, these factor-responsive human leukemia cell lines represent extremely useful model systems for the analysis of cytokine effects on hematopoietic cells. The cytokine response profiles of the individual cell lines provide guidelines for the selection of the appropriate cell culture for such experiments.

Expression of HOX Genes in Acute Leukemia Cell Lines with and without MLL Translocations

In primary cells from acute leukemia patients, expression of the genes MEIS1, HOXA5, HOXA7 and HOXA9 has been reported to be correlated with the occurrence of MLL translocations. It was our aim to find out whether MLL mutant (MLLmu) and MLL wild-type (MLLwt) acute leukemia-derived cell lines might likewise be discriminated on the basis of HOX gene expression. Southern blot analysis, performed to verify the MLL status of the cells, showed that NOMO-1 was the only cell line not tested previously carrying a rearranged MLL gene. Fluorescence in situ hybridization analysis demonstrated that this cell line exhibited a reciprocal t(9;11)(q23;p22). Sequencing of RT-PCR products thereof identified unique MLL exon 10/AF-9 exon 5 fusion transcripts. We divided the acute leukemia-derived cell lines (n = 37) according to the results of Southern blot analysis into MLLmu (n = 19) and MLLwt (n = 18). Expression of HOX genes was then analyzed by applying reverse transcriptase-polymerase chain reaction, Northern and Western blot analyses. Acute myeloid leukemia (AML) cell lines expressed the HOX genes significantly more often than acute lymphoblastic (ALL) cell lines. In ALL, cells with MLL translocations expressed the genes 4 times more often than MLLwt cells. Most distinct was the correlation between MLL status and MEIS1 expression in ALL-derived cell lines: 8/8 MLLmu, but 0/10 MLLwt cell lines expressed MEIS1. Northern and Western blot analysis confirmed that also HOXA9 and FLT3 were significantly more often and stronger expressed in MLLmu than in MLLwt ALL cell lines. These results suggest that MLL aberrations may regulate MEIS1 and HOXA9 gene expression in ALL-derived cell lines, while AML-derived cell lines express these genes independently of the MLL status.

HLXB9 activates IL6 in Hodgkin lymphoma cell lines and is regulated by PI3K signalling involving E2F3.

Multiple cytokines are secreted by Hodgkin lymphoma (HL) cells, notably interleukin-6 (IL6), which is believed to play a significant pathobiological role in this and certain other tumors. Previous work on prostate carcinoma cells has shown that IL6 expression is activated therein by the homeodomain protein GBX2, which we found to be absent in HL cells. Instead, we observed expression of a closely related gene, HLXB9, albeit restricted to HL cells coexpressing IL6. Treatment of HL cell lines with antisense-oligonucleotides directed against HLXB9, forced expression of recombinant HLXB9, and analysis of reporter gene constructs containing IL6 promoter sequences all confirmed the potential of HLXB9 to drive expression of IL6. Chromosomal rearrangements of the HLXB9 locus at 7q36 were not detected in HL cells unlike AML subsets expressing HLXB9. However, inhibition of certain signal transduction pathways revealed that the phosphatidylinositol 3 kinase (PI3K) pathway contributes to HLXB9 expression. AKT/phospho-AKT analysis revealed constitutively active PI3K signalling in HL cell lines. Downstream analysis of PI3K revealed that E2F3 may mediate activation of HLXB9. Taken together, our data show that the PI3K signalling pathway in HL cells is constitutively activated and promotes HLXB9 expression, probably via E2F3, thereby enhancing malignant expression of IL6.

SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

BackgroundSET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene.ResultsOf 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF- Iβ)-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF- I isoforms revealed that the cell lines also expressed TAF- Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.ConclusionCell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

SOCS2: inhibitor of JAK2V617F-mediated signal transduction

Janus kinase 2 (JAK2)V617F-activating mutations (JAK2mu) occur in myeloproliferative disorders (MPDs) and myelodysplastic syndromes (MDSs). Cell lines MB-02, MUTZ-8, SET-2 and UKE-1 carry JAK2V617F and derive from patients with MPD/MDS histories. Challenging the consensus that expression of JAK2V617F is the sole precondition for cytokine independence in class I cytokine receptor-positive cells, two of four of the JAK2mu cell lines were growth factor-dependent. These cell lines resembled JAK2wt cells regarding JAK2/STAT5 activation: cytokine deprivation effected dephosphorylation, whereas erythropoetin or granulocyte colony-stimulating factor induced phosphorylation of JAK2 and STAT5. Cytokine independence correlated with low expression and cytokine dependence with high expression of the JAK/STAT pathway inhibitor suppressor of cytokine signaling 2 (SOCS2) suggesting a two-step mechanism for cytokine independence of MPD cells: (i) activation of the oncogene JAK2V617F and (ii) inactivation of the tumor suppressor gene SOCS2. Confirming that SOCS2 operates as a negative JAK2V617F regulator, SOCS2 knockdown induced constitutive STAT5 phosphorylation in JAK2mu cells. CpG island hypermethylation is reported to promote SOCS gene silencing in malignant diseases. Accordingly, in one of two cytokine-independent cell lines and in two of seven MPD patients, we found SOCS2 hypermethylation associated with reduced promoter access to transcription factors. Our results provide solid evidence that SOCS2 epigenetic downregulation might be an important second step in the genesis of cytokine-independent MPD clones.