Bcl-6 May Be a Survival Factor in Pre-B Cell Ph+ Blast Crisis Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4536-4536
Author(s):  
Ana Batlle ◽  
Eric W. Lam ◽  
Simon D. Wagner
Keyword(s):  
Transcription Factor ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Negative Control ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Mammalian Expression ◽  
Survival Factor

Abstract The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression plasmid bearing a dominant negative STAT5 reduced Bcl-6 expression in Z119 implying that activated STAT5 promotes Bcl-6 expression in this cell line, but also that STAT5 cannot be the main target of Bcr-Abl because Imatinib and dominant negative STAT5 have opposing effects. Next we considered the possibility that phosphorylation status of the transcription factor FoxO3a, which has been shown to bind to the Bcl-6 promoter, correlated with Bcl-6 expression. We found that decreased phosphorylation of Foxo3a, activating this transcription factor, was highly associated with increased Bcl-6 expression suggesting that it is an significant Bcr-Abl target. Next we wished to find out the functional implications of increased Bcl-6 expression. We found that a low concentration of Imatinib (0.25μM) was sufficient to induce Bcl-6 in BV173 and Z119, and caused death of these cell lines over several days. Next we combined Imatinib with a previously reported peptide antagonist of the Bcl-6/SMRT co-repressor interaction. Neither this peptide nor a mutated negative control peptide had an effect on cell numbers or apoptosis over 48 hours of culture when used alone. However, when combined with Imatinib the wild-type peptide, but not the control, reduced cell growth in Z119. We conclude that Bcl-6 may be a survival factor in Ph+ lymphoid blast crisis cell lines following treatment with Imatinib and that specific treatments to abrogate Bcl-6 function may find a place in the treatment of lymphoid blast crisis of CML.

IKZF1 (Ikaros) Deletions Are a Hallmark of BCR-ABL1 Positive Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 721-721
Author(s):  
Charles G. Mullighan ◽  
Christopher B. Miller ◽  
Letha A. Phillips ◽  
James Dalton ◽  
Jing Ma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Cell Lines ◽  
Progenitor Cell ◽  
Blast Crisis ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Snp Arrays ◽  
Progenitor Cell Line

Abstract Using high-resolution SNP arrays and genomic resequencing, we recently reported deletions, translocations, and mutations involving regulators of B cell development in 40% of pediatric B-progenitor ALL (Nature2007;446:758). The most frequently involved genes were PAX5, EBF1, IKZF1 (Ikaros), IKZF3 (Aiolos) and LEF1. Ikaros is a transcription factor required for normal lymphoid development and acts as a tumor suppressor in mice. Focal deletions of IKZF1 were observed in 7 B-progenitor ALL cases, suggesting that the previously reported expression of dominant-negative, non-DNA binding Ikaros isoforms may be caused by genomic IKZF1 abnormalities. We have now extended the analysis to 283 pediatric ALL cases, including B-progenitor ALL with high hyperdiploidy (N=39), hypodiploidy (N=10), rearrangement of MLL (N=23), TCF3-PBX1 (N=17), ETV6-RUNX1 (N=48) and BCR-ABL1 (N=21), as well as cases with low hyperdiploid, normal or miscellaneous cytogenetics (N=75), and T-lineage ALL (N=50). We also examined 22 adult BCR-ABL1 positive ALL cases, 37 acute leukemia cell lines and 49 samples from 23 chronic myeloid leukemia (CML) cases at various stages of disease, including 15 with matched blast crisis samples (12 myeloid, 3 lymphoid). All samples were examined with 500,000 feature Affymetrix SNP arrays (250k Sty and Nsp). The 50k Hind and Xba arrays were also used in 252 ALL cases. Sixty-two (20.3%) ALL cases harboured IKZF1 deletions, including 36 of 43 (83.7%) BCR-ABL1 positive B-ALL cases (76.2% of 21 childhood cases, and 90.9% of 22 adult cases). The deletions were limited to IKZF1 in 25 BCR-ABL1 ALL cases, and in 19 cases deleted an internal subset of IKZF1 exons, most commonly 3–6 (Δ3–6). Remarkably, chronic phase CML samples lacked evidence of IKZF1 deletion, whereas four of 15 matched CML blast crisis samples (66% of lymphoid and 17% of myeloid) had acquired an IKZF1 deletion. The IKZF1 Δ3–6 deletion was also detected in the BCR-ABL1 B-progenitor cell lines BV173, OP1 and SUP-B15, the Δ1–6 deletion in the MYC-IGH/BCL2-IGH B-progenitor cell line 380, and Δ1–7 in the BCR-ABL1 B-progenitor cell line TOM-1. RT-PCR analysis for IKZF1 transcripts demonstrated complete concordance between the extent of IKZF1 deletion and the expression of aberrant Ikaros transcripts lacking internal exons. Importantly, on quantitative RT-PCR analysis and western blotting, expression of the dominant-negative Ik6 transcript and protein, which lacks exons 3–6, was exclusively observed in those cases with IKZF1 Δ3–6, demonstrating that the Ik6 transcript is the result of a specific genetic lesions and not alternative splicing of wild-type IKZF1. Lastly, sequence analysis of the IKZF1 Δ3–6 breakpoints indicated that the deletions arise from aberrant activity of RAG-mediated V(D)J recombination. Taken together, these data demonstrate that deletion of IKZF1, resulting in either haploinsufficiency or the expression of a dominant negative form of the transcription factor, is a central event in the pathogenesis of both pediatric and adult BCR-ABL1 B-progenitor ALL.

scholarly journals Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1931-1941 ◽  
Author(s):  
A Neubauer ◽  
A Fiebeler ◽  
DK Graham ◽  
JP O'Bryan ◽  
CA Schmidt ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Intracellular Signaling ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Tissue ◽  
Blood Leukocytes

Abstract We previously reported the cloning, and characterization of a receptor tyrosine kinase, axl, from two patients with chronic myelogenous leukemia. Herein, we describe the expression pattern of axl in normal and malignant hematopoietic tissue axl message is detected in normal human bone marrow but not significantly in normal blood leukocytes. Cell separation experiments showed that axl is expressed in hematopoietic CD34+ progenitor and marrow stromal cells, at low levels in peripheral monocytes, but not in lymphocytes or granulocytes. Consistent with the normal pattern of axl expression, axl RNA was found predominantly in diseases of the myeloid lineage: 39 of 66 (59%) patients with myeloproliferative disorders (acute myeloid leukemia, chronic myeloid leukemia (CML) in chronic phase, CML in myeloid blast crisis, and myelodysplasia) showed significant axl transcription, as compared with 1 of 45 (2%) lymphoid leukemias (chronic lymphocytic leukemia, acute lymphocytic leukemia, and CML in lymphoid blast crisis). Treatment of K562 cells with the phorbol ester, 12-O- tetradecanoylphorbol-13-acetate (TPA), administration of interferon alpha (IFN alpha) to normal monocytes, and treatment of U937 cells with TPA and IFN tau significantly induced axl expression, supporting a role for this kinase in the intracellular signaling of myeloid cells through a variety of biochemical pathways. These results suggest that the axl kinase may be operative in normal and malignant myeloid biology.

scholarly journals The receptor tyrosine kinase p185HER2 is expressed on a subset of B- lymphoid blasts from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1916-1923 ◽  
Author(s):  
HJ Buhring ◽  
I Sures ◽  
B Jallal ◽  
FU Weiss ◽  
FW Busch ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Lymphoblastic Leukemia ◽  
Blast Crisis ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
B Lymphoid

The class I receptor tyrosine kinase (RTK) HER2 is an oncoprotein that is frequently involved in the pathogenesis of tumors of epithelial origin. Here we report mRNA expression in peripheral blood and bone marrow cells from healthy donors in hematopoietic cell lines and leukemic blasts from patients with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), chronic lymphoblastic leukemia (CLL), and chronic myeloid leukemia (CML). However, cell surface expression of HER2 protein (p185HER2) was found exclusively on a subset of leukemic cells of the B-lymphoblastic lineage. p185HER2 expression was found on blasts in 2 of 15 samples from infants, 9 of 19 samples from adult patients with C-ALL (CD19+CD10+), and 1 of 2 samples from patients with pro-B ALL (CD19+CD10-), whereas none of the leukemic cells from patients with AML (0/30), T-ALL (0/7), CLL (0/5) (CD19+CD5+), or CML in chronic and accelerated phase (0/5) or in blast crisis with myeloid differentiation (0/14) were positive for p185HER2. However, cells from 3 of 4 patients with CML in B-lymphoid blast crisis (CD19+CD10+) expressed high levels of p185HER2, which was also found on the surface of the CML-derived B-cell lines BV-173 and Nalm-1. Our study shows p185HER2 expression on malignant cells of hematopoietic origin for the first time. Aberrant expression of this oncogenic receptor tyrosine kinase in hematopoietic cell types may be an oncogenic event contributing to the development of a subset of B- lymphoblastic leukemias.

Jak2: A Potential Therapeutic Target for Chronic Myelogenous Leukemia (CML).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2121-2121
Author(s):  
Ajoy K. Samanta ◽  
Hui Lin ◽  
Tong Sun ◽  
Hagop Kantarjian ◽  
Ralph B. Arlinghaus
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Imatinib Treatment ◽  
Potential Therapeutic Target ◽  
Imatinib Resistant

Abstract In most CML patients Bcr-Abl, a constitutively active tyrosine kinase derived from the Philadelphia chromosome, is highly expressed and is the causative factor in most CML patients. Imatinib mesylate, an inhibitor of the Bcr-Abl kinase, is a very effective drug for treatment of CML. However in some CML patients, drug resistance develops and the patients relapse. Thus, alternative drug targets need to be identified. We have shown that Bcr-Abl activates its downstream target, the Jak2 tyrosine kinase, leading to the enhancement of c-Myc expression (Xie et al. Oncogene21: 7137, 2002; Samanta et al. Cancer Res.66: 6468, 2006). Our recent studies showed that Bcr-Abl activated the transcriptional factor NF-kB through Jak2, which in turn activated c-Myc transcription. Jak2 also activated Akt, which increased c-Myc protein levels by inhibiting GSK3. Addition of AG490, an inhibitor of the Jak2 kinase, prevented enhanced expression of c-Myc and caused induction of apoptosis in BCR-ABL+ leukemia cells. Immunoprecipitation experiments showed that Bcr-Abl is associated with a cluster of signaling proteins including Jak2, Gab2, Akt and GSK3b. Treatment of CML cell lines and mouse BCR-ABL+ 32D cells (myeloid lineage) with the either Jak2 siRNA or the Jak2 kinase inhibitor AG490 caused inhibition of pTyr Gab2 formation, pSer Akt formation and the activation of NFkB. Of interest, treatment of BCR-ABL+ 32 D cells with IL-3 reversed the apoptotic effects of imatinib by activation of Jak2 even though Bcr-Abl was inhibited. Importantly, mouse BaF3 hematopoietic cells expressing the T315I and E255K imatinib-resistant mutants of BCR-ABL underwent apoptosis upon exposure to either the Jak2 inhibitor AG490 or siRNA for Jak2, yet were resistant to imatinib. Cells from a number of CML patients (including six chronic phase, one accelerated phase, and two blast crisis patients who failed imatinib treatment) were induced to enter apoptosis upon treatment with AG490, whereas normal samples were not affected by AG490. Further analysis of imatinib resistant Bcr-Abl cell lines showed that transfection of the cells with Jak2 specific siRNA or by treating the cells with AG490 reduced levels of pLyn, pAkt, c-Myc and pGSK3 level compared to untreated cells. Transfection of Lyn specific siRNA into K562 and 32Dp210 cells resulted in down-regulation of pGab2, pAkt, pGsk3 and c-Myc, but did not alter pJak2 levels; this result indicates that pLyn is downstream of Jak2 but upstream of Gab2, pAkt, pGSK3 in BCR-ABL+ leukemia cells. We hypothesize that Jak2 activation of Lyn tyrosine kinase in BCR-ABL+ leukemia cells leads to tyrosine phosphorylation of the YxxM motif of Gab2, which activates the PI-3 kinase-Akt pathway. In conclusion, since inactivation of Jak2 inhibits many of the critical oncogenic targets of Bcr-Abl (resulting in apoptosis induction), we propose that Jak2 is a potential therapeutic target for CML, in both imatinib sensitive and imatinib resistant patients.

Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 324-324
Author(s):  
Xiaoyan Jiang ◽  
Donna Forrest ◽  
Franck Nicolini ◽  
Karen Lambie ◽  
Kyi Min Saw ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Clinical Responses ◽  
Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P<0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and >50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P<0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

Targeting Autophagy Potentiates Imatinib-Induced Cell Death in Philadelphia Positive Cells Including Primary CML Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1070-1070 ◽  
Author(s):  
Cristian Bellodi ◽  
Maria Rosa Lidonnici ◽  
Ashley Hamilton ◽  
Gudmundur V Helgason ◽  
Angela R Soliera ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Competitive Inhibitor ◽  
Myelogenous Leukemia ◽  
Stem Cell Population ◽  
Therapeutic Effects

Abstract Imatinib mesylate (IM), a potent ATP-competitive inhibitor of the BCR/ABL tyrosine kinase, has become standard therapy for patients with chronic myelogenous leukemia (CML). However, the main limitations of IM- and second generation tyrosine kinase inhibitor (TKI)-based therapy are the insurgence of resistance in patients and the intrinsic refractoriness of primitive Philadelphia-positive stem cells. Therefore, there is the need to develop new therapeutic approaches that, in combination with TKI, might be more effective in targeting the stem cell population and preventing the outgrowth of TKI-resistant CML cells. TKI-induced elimination of BCR/ABL-dependent intracellular signals is known to trigger apoptosis, but it is unclear whether this also activates additional cell death and/or survival pathways. We show that IM treatment induces autophagy in CML blast crisis cell lines, CML primary cells and p210BCR/ABL-expressing 32Dcl3 (32D) myeloid precursor cells, but not in 32D cells expressing v-Src or the IM-resistant T315I p210BCR/ABL mutant. IM-induced autophagy does not involve c-Abl, as it is also observed in cells co-expressing p210BCR/ABL and the IM-resistant T315I c-Abl mutant. Induction of autophagy is associated with endoplasmic reticulum-stress and is suppressed by depletion of intracellular calcium. By contrast, ectopic Bcl-2 expression does not block IM-induced autophagy. Suppression of autophagy by pharmacological inhibitors or siRNA-mediated knockdown of essential autophagy genes enhances cell death induced by IM in cell lines and primary CML cells, demonstrating that induction of autophagy has a pro-survival effect. Critically, the combination of TKI with autophagy inhibitors results in near complete elimination of phenotypically (CD34+38−) and functionally (colony forming cells) defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKI in the treatment of CML.

CML Patients Show Sperm Alterations At Diagnosis That Are Not Improved On Tyrosine Kinase Inhibitor Treatment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1669-1669
Author(s):  
Franck E. Nicolini ◽  
Françoise Huguet ◽  
Hélène Labussière-Wallet ◽  
Yann Guillermin ◽  
Madeleine Etienne ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Control Group ◽  
Semen Volume ◽  
The Impact ◽  
Williams Test

Abstract Abstract 1669 Most epidemiologic studies performed in chronic myelogenous leukemia (CML) relate that the disease occurs preferentially in males with a sex ratio of ∼1.2. In addition, CML can be diagnosed in young adults and masculine fertility is a matter of concern, particularly because tyrosine kinase inhibitors (TKI) may impact on spermatogenesis by a selective inhibition of Src kinases, PDGF-R and c-kit. Sperm cryopreservation is recommended by some authors at diagnosis in males that would expect to have children later on. In a retrospective analysis we have analysed the spermograms of 62 chronic phase (CP) and 2 onset blast crisis (BC) CML males referred to our 3 centres between 2001 and 2012, collected at diagnosis before TKI treatment, and we have compared the results obtained to those of 15 healthy volunteer donors from the cryopreservation bank database, after informed consent. In 10 patients we could collect some data for patients being on imatinib mesylate (IM). CML patients had a median age of 31 (16–48) years, significantly younger than that in the control group of healthy donors: 37 (34–45) years (p=0.001). Sokal scores were 24% high, 27% intermediate and 49% low for evaluable patients (13 patients unknown or not available). The median BCR-ABLIS value at diagnosis was 77.65%. Patients had a median duration of 26 (0–38) days of hydroxyurea prior to commencing any TKI and 65% of evaluable patients had HU before TKI. None of the patients got interferon prior to TKI. The semen cryopreservation was performed within a median of 10 (2–102) days after CML diagnosis and after a median abstinence of 5 (0.5–30) days. The median volume of semen obtained in CML patients was 2.95 (0.5–14.9) ml and 3 (1.4–5.3) ml for normal donors (p=0.3). Williams test showed 72 (0–87)% of necrospermia in patients versus 18 (4–32)% in donors (p=0.00003). The median number of spermatozoa obtained was not different in patients [46 (0.03–200) 106/ml] than that in donors [74 (19.2–253) 106/ml] (p=0.24), as well as the number of spermatozoa per ejaculate observed (p=0.49). The motility of spermatozoa at 30 minutes after collection was not different between patients (median = 47.5%) and donors (median = 50%) (p=0.12), however higher numbers of atypical spermatozoa were observed in patients [median = 77.5 (16–100)%] rather than in donors [median = 45% (22–89)%], p=0.008, and the multiple abnormalities index (MAI) was significantly higher in patients [median = 1.99 (1.14–2.7)] than that in donors [median = 1.33 (1.09–1.55)], p=0.00006. There was no correlation between age at diagnosis, Sokal index and the number of spermatozoa per ml obtained (p=0.7 and 0.21 respectively). Ten CP CML patients had spermograms after a median of 1440 (9–1456) days of IM treatment and the results obtained were compared to i) the results of each individual patient at CP diagnosis and ii) to the results of healthy comparators. In comparison to the characteristics observed at diagnosis, the semen volume (median = 3.1 ml), Williams test (median = 65%), the motility at 30 minutes (median = 37.5%) and the MAI (median = 1.71) were not different (p=ns for all), however, the numbers of spermatozoa (median = 14.9 106/ml and = 37.05 ml per ejaculate) collected on IM were significantly lower (p=0.014 and p=0.045 respectively). The different parameters evaluated on IM were compared to those of normal controls and showed significant alterations. The semen volume was not different (p=0.94), neither the motility of spermatozoa (p=0.24), but the Williams test was highly perturbed on IM [median 65 (24–79)% versus 18 (4–32)% in donors] p=0.00003, as well as the numbers of spermatozoa as 106 per ml, collected on IM [median 14.9 (0.67–179)) versus normal [74 (19.2–253)], p=0.0036 or as 106 per ejaculate collected on IM [median 37.5 (2.68–572.8)) versus normal [149 (30–535.3)], p=0.026. Atypical forms were significantly more abundant on IM [median = 80 (68–90)%] versus healthy controls [median = 45% (22–89)], p=0.0058. Finally, the MAI was severely altered on IM [median = 1.71 (1.61–1.98)] versus normal individuals [median = 1.33 (1.09–1.55)], p=0.00013. In conclusion, this work demonstrates the existence of significant sperm alterations in young males with CML at diagnosis of undetermined origin, prior to any treatment. These alterations persist on IM treatment and little is know about the impact of second generation TKI. Thus the most appropriate approach remains a matter of debate in thus setting. Disclosures: Nicolini: Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Huguet:Novartis, BMS: Speakers Bureau. Michallet:Novartis, Pfizer, Teva, Genzyme, Janssen Cilag, BMS, Merck, Pfizer, Gilead, Alexion: Consultancy, Speakers Bureau. Etienne:Novartis, Pfizer, speaker for Novartis, BMS: Consultancy.

SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 4130-4138 ◽  
Author(s):  
Jean-François Honorat ◽  
Ashraf Ragab ◽  
Laurence Lamant ◽  
Georges Delsol ◽  
Jeannie Ragab-Thomas
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Tyrosine Phosphatase ◽  
Inverse Correlation ◽  
Large Cell ◽  
Tissue Microarrays ◽  
Negative Regulator ◽  
3T3 Fibroblasts ◽  
Alk Positive

Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

The Biology of CML Blast Crisis

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 384-391 ◽  
Author(s):  
Jerald P. Radich
Keyword(s):  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Allogeneic Transplantation ◽  
Cancer Evolution ◽  
Advanced Phase ◽  
History Of ◽  
Clinical Dilemma ◽  
Predictive Assays

Abstract The natural history of chronic myeloid leukemia (CML) progresses from a relatively benign chronic phase into a fatal blast crisis, which resembles acute leukemia, but is incurable by chemotherapy. Fortunately, the progression can usually be blocked by tyrosine kinase therapy or allogeneic transplantation. The seemingly stereotypical march of progression involves changes in genetic instability and DNA repair, proliferation, differentiation, and apoptosis, and thus may serve as a unique model of cancer evolution and progression. Given that all treatments work much better in chronic-phase than advanced-phase disease, the clinical dilemma is predicting and detecting patients bound to evolve into advanced disease. This is especially important in the age of tyrosine kinase inhibition (TKI) therapy. The purpose of this review is to address the biology of blast crisis in the age of tyrosine kinase therapy, with an emphasis on what genes or pathways may be future targets of predictive assays or treatments of progression.

scholarly journals Propagation of human blastic myeloid leukemias in the SCID mouse

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2089-2098 ◽  
Author(s):  
CL Sawyers ◽  
ML Gishizky ◽  
S Quan ◽  
DW Golde ◽  
ON Witte
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Scid Mouse ◽  
Lymphoblastic Leukemia ◽  
Blast Crisis ◽  
Late Phase ◽  
Severe Combined Immunodeficiency ◽  
Chronic Phase ◽  
Myeloid Leukemias

Existing in vitro culture technology does not permit the routine propagation of most human myeloid leukemias. Previous work has shown the usefulness of mice with severe combined immunodeficiency (SCID) for the growth of human lymphoblastic leukemia. We show here that human myeloid cell lines and bone marrow samples from patients with acute myeloid leukemia (AML) and blast crisis of chronic myeloid leukemia (CML) also grow in SCID mice. Human AML or CML cell lines (three of three lines tested) grew in the bone marrow and peripheral blood of the mice after intravenous (IV) inoculation in a pattern closely resembling human AML. To define the best conditions for the growth of primary human myeloid leukemia cells, samples were transplanted into mice at several alternative sites. Using flow cytometry and Southern analysis, mice were analyzed at defined intervals up to 36 weeks after transplantation for the presence of human cells in various tissues. For four of four patients with AML and two of two patients with blast crisis of CML, myeloblasts grew locally at the site of implantation and were detected in the murine hematopoietic tissues. In contrast, marrow implants from patients in the chronic phase of CML (six patients) showed infrequent and limited myeloid growth in the mice. These findings demonstrate that the SCID mouse is a reproducible system for the propagation of blastic human myeloid leukemias. The differential growth of early- versus late-phase CML suggests that the SCID mouse may be a useful assay for identifying biologically aggressive leukemias early in their clinical presentation.

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