Erythropoietin Regulates GATA-1 Function and Erythroid Differentiation by a PKD-HDAC5 Signal Transduction Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 85-85
Author(s):  
Lorrie L. Delehanty ◽  
Adam N. Goldfarb
Keyword(s):  
Cell Death ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Class Ii ◽  
Dependent Manner ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Nadir Point ◽  
Shrna Knockdown

Abstract Abstract 85 How erythropoietin (Epo) signaling promotes erythroid differentiation remains unclear. Epo is known to regulate the function of the master regulator of erythroid gene transcription, GATA-1. Using murine proerythroblasts engineered with a conditional GATA-1-ER fusion, G1ER cells, Gregory et al. showed that GATA-1 induction of erythroid differentiation required signaling by Epo; in cultures lacking Epo, activation of GATA-1 caused cell death without differentiation (Blood 94:87-96, 1999). Although several models have been proposed for Epo regulation of GATA-1, no mechanism has been established. While analyzing PKC isozymes regulated by Epo and iron in primary human erythroblasts, our lab identified PKCμ , aka PKD, as activated in a dosage-dependent manner by Epo. Subsequent studies in Epo-starved G1ER cells, and in primary human progenitors, confirmed direct Epo induction of PKD phosphorylation on Ser744/748. A major PKD pathway involves its phosphorylation of class II HDACs (4, 5, 7 and 9), leading to release of HDAC-bound transcription factors (e.g. MEF2) from tonic repression. Watamoto et al. previously identified GATA-1 as a class II HDAC regulated factor, displaying physical and functional interaction with HDAC5 (Oncogene 22:9176-9184, 2003). Accordingly, we employed G1ER cells to determine whether Epo signaling to GATA-1 involved the PKD-HDAC pathway. In G1ER cells cultured in stem cell factor (SCF) minus Epo, endogenous HDAC5, but not HDAC4, co-immunoprecipitated with GATA-1. Epo stimulation induced dissociation of the HDAC5-GATA-1 complex, without affecting levels of either factor. The function of erythroid PKD-HDAC signaling was addressed by pharmacologic and shRNA approaches. Initial experiments tested whether inhibition of HDAC activity could substitute for Epo signaling in G1ER cells undergoing estradiol activation of GATA-1-ER. As described, GATA-1 activation in cells grown in SCF without Epo caused >90% cell death with minimal hemoglobinization after 48 hours. Addition of the HDAC inhibitor SAHA completely rescued viability and partially restored hemoglobinization of cells undergoing GATA-1 activation in the absence of Epo. Along similar lines, shRNA knockdown of HDAC5, but not HDAC4, significantly enhanced viability and hemoglobinization of G1ER cells undergoing GATA-1 activation under limiting Epo concentrations (0.05 U/ml). Conversely, shRNA knockdown of PKD3 impaired hemoglobinization of cells undergoing GATA-1 activation in the presence of Epo. Further implicating PKD in erythropoiesis, differentiation of G1ER cells was blocked by the kinase inhibitor Gö6976, which targets PKC and PKD, but not by the related compound Gö6983, which targets only PKC. Similar results were obtained with primary human progenitors, in which the PKD inhibitor Gö6976 but not the classical PKC inhibitor Gö6983 inhibited erythroid differentiation. In vivo roles of PKD-HDAC signaling in erythropoiesis were addressed by studying HDAC5-/- mice. In contrast to wild type counterparts, adult HDAC5-/- mice showed elevated steady state hematocrits (56.2 ± 0.8 vs 39.3 ± 1.4, P < 0.0001), resulting from increased MCVs (68.8 ± 0.7 vs 48.2 ± 0.1, P < 0.0001). In response to PHZ-induced hemolytic anemia, HDAC5-/- mice showed higher nadir RBC counts (5.0 ± 0.3 vs 3.2 ± 0.2, P < 0.001) and displayed an unexpected increase in Hb levels at the nadir point (15.9 ± 0.8 vs 8.6 ± 0.5, P < 0.0001). Spleens and livers obtained post recovery showed increased iron deposition in HDAC5-/- mice, consistent with increased net red cell turnover. To determine whether the observed abnormalities were cell-intrinsic, we sorted CD71++ Ter119- erythroid progenitors from the marrows of adult HDAC5-/- and wild type mice. The HDAC5-/- progenitors, but not wild type counterparts, displayed erythroid differentiation, manifested by Ter119 upregulation, in medium with no or low Epo (0.01-0.05 U/ml). Our data thus implicate PKD-HDAC signaling in Epo regulation of GATA-1 function and thereby provide a mechanistic basis for an instructional function of the Epo receptor. In addition, activation of PKD in non-erythroid cells could potentially explain some of the poorly-understood clinical complications of Epo therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals KIT with D816 Mutations Cooperates with CBFB-MYH11 for Leukemogenesis in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 867-867
Author(s):  
Ling Zhao ◽  
J. Joseph Melenhorst ◽  
Lemlem Alemu ◽  
Stacie Anderson ◽  
Martha Kirby ◽  
...  
Keyword(s):  
Cell Death ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Retroviral Vectors ◽  
Mapk Signaling ◽  
Leukemia Cells ◽  
Erythroid Progenitors ◽  
Kit Mutation

Abstract Abstract 867 KIT mutations are the most common secondary mutations in inv(16) AML patients (10–45%), and their presence may suggest poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis and to investigate the underlying mechanism. Here, we transduced bone marrow (BM) cells from wild type (WT) and conditional Cbfb-MYH11 knock-in (Cbfb+/56m; Tg(Mx1-Cre)) mice with retroviral vectors carrying wildtype (WT) or mutant (activation mutations D816V or D816Y) KIT variants. In colony forming assays, KIT (WT or D816 mutants) transduction led to significantly fewer colonies (> 7 fold decrease) from WT BM cells, whereas the Cbfb+/56m; Tg(Mx1-Cre) BM cells were only mildly affected (1.6 fold decrease in colony numbers). Further analysis of transduced BM cells indicated that KIT transduction significantly (p<0.05) increased cell death in transduced Lin− BM cells (both Cbfb+/56m; Tg(Mx1-Cre) and WT), as compared to untransduced Lin− BM cells, which could explain the decreased total colony numbers. Analysis of WT Lin− BM cells transduced with WT KIT and D816 mutants showed similar massive cell death (87% (wt); 87% (D816Y); 94% (D816V)(N=4 for each). On the other hand, in transduced Cbfb+/56m; Tg(Mx1-Cre) Lin− BM cells, the cell death rates were 74.3% (wt), 55.2% (D816Y) and 84.5% (D816V)(N=4 for each). This difference in the level of cell death could explain the differential effects of KIT on colony formation from transduced Cbfb+/56m; Tg(Mx1-Cre) BM cells vs. WT BM cells. These results also suggest that BM cells expressing Cbfb-MYH11 are more resistant to the toxic effects of KIT than WT BM cells. Moreover, more mixed-lineage (CFU-GEMM) and fewer erythroid (BFU-E) colonies were obtained from Cbfb+/56m; Tg(Mx1-Cre) BM cells transduced with D816V/Y KIT than those transduced with WT KIT, suggesting differentiation defects in early myeloid and erythroid progenitors were induced by the mutant KIT. We then transplanted the transduced BM cells and found that 60% and 80% of mice transplanted with Cbfb+/56m; Tg(Mx1-Cre) BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, while none of the control mice did. Results from limiting dilution transplantations using multiple donor leukemia cells (N=3) showed that mice transplanted with as little as 10 cells died from leukemia within two month, while mice transplanted with 106Cbfb+/56m; Tg(Mx1-Cre) leukemia cells died around two months. We also found a significant increase of mitotic cells in Cbfb+/56m; Tg(Mx1-Cre) leukemic spleen cells that carried the KIT mutations. These data indicate that the KIT D816 mutations not only facilitate the transformation of Cbfb+/56m; Tg(Mx1-Cre) BM cells to leukemia cells, but also help maintain these leukemia cells with higher leukemia initiating cells and proliferation. We next explored the response of this aggressive leukemia to a novel small molecule, the kinase inhibitor PKC412, and found that Cbfb+/56m; Tg(Mx1-Cre) leukemia cells carrying KIT D816 mutations were sensitive to this kinase inhibitor, with significantly less survival than leukemia cells without mutant KIT and cells treated by vehicle only after overnight treatment. Signaling pathway analysis of these leukemia cells suggested that Stat3 and P44/42 MAPK signaling, which has been reported to be activated in cancer cells and is involved in cell proliferation, might be imported for these leukemia cells. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis and show that acute myeloid leukemia cells carrying the inv(16) fusion gene and an activating KIT mutation respond to the small molecule PKC412. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Active elimination of intestinal cells drives oncogenic growth in organoids

2020 ◽  
Author(s):  
Ana Krotenberg Garcia ◽  
Arianna Fumagalli ◽  
Huy Quang Le ◽  
Owen J. Sansom ◽  
Jacco van Rheenen ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Death ◽  
Cancer Cells ◽  
Cell Fate ◽  
Crucial Role ◽  
Intestinal Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Cell Competition ◽  
Type Population

AbstractCompetitive cell-interactions play a crucial role in quality control during development and homeostasis. Here we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell fate transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. JNK signalling is activated in competing cells and is required for cell fate change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell fate change in a JNK dependent manner.

Anti-Leukemia and Multiple Myeloma Selective Activity of CXCR4 Antagonist 4F-Benzoyl-TN14003 Involves Apoptotic Death Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3857-3857
Author(s):  
Katia Beider ◽  
Michal Begin ◽  
Michal Abraham ◽  
Hanna Wald ◽  
Ido Weiss ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Jurkat Cells ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Hl60 Cells

Abstract Abstract 3857 Poster Board III-793 The chemokine receptor CXCR4 and its ligand CXCL12 are involved in the development and progression of a diverse number of hematological malignancies, including leukemia, lymphoma and multiple myeloma (MM). Binding CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways and effector molecules that regulate cell chemotaxis, adhesion, survival, apoptosis and proliferation. It was previously shown that CXCR4 signaling can directly induce caspase-independent cell apoptosis through the interaction with the HIV gp120 envelope protein. In the present study we investigated the effect of CXCR4 specific antagonists 4F-benzoyl-TN14003 (T140) and AMD3100 on the survival and proliferation of different human hematological cancer cells. Here, we demonstrate that T140, but not AMD3100, exhibits preferential cytotoxicity towards malignant cells of hematopoietic origin, as compared to primary normal cells or solid prostate and breast tumor cells. The in vitro treatment with T140, but not with AMD3100, significantly decreased the number of viable chronic myeloid leukemia K562 cells, acute T cell leukemia Jurkat cells, acute promyelocytic leukemia NB4 and HL60 cells, and four different MM cell lines (U266, NCI-H929, RPMI8226 and ARH77), demonstrating the highest sensitivity to T140 (p<0.01). Notably, T140 inhibited the growth of freshly isolated leukemia and MM cells obtained from consenting patients. T140 inhibits the growth of MM and leukemic cells by inducing their apoptotic cell death. The apoptotic changes in the cells were associated with morphological changes, phosphatidylserine externalization, sub-G1 arrest, DNA double-stranded breaks, decrease in mitochondrial membrane potential, release of cytochrome c, and caspase 3 activation. The important role of CXCR4 in T140-mediated cell death was confirmed by demonstrating that CXCR4 over-expression in NB4 and K562 cells increased their sensitivity to T140. Furthermore, pretreatment of NB4 and HL60 cells with AMD3100 abolishes the effect of T140 on these cells, indicating the involvement of CXCR4 in T140-induced apoptosis. Importantly, the combination with novel anti-myeloma agent bortezomib significantly augments anti-myeloma activity of T140. The anti leukemic and MM effect of T140 was confirmed in xenograft in vivo tumor models. Subcutaneous (s.c.) or intra-peritoneal (i.p.) injections of T140 (100 or 300 mcg/mouse) significantly reduced, in a dose-dependent manner, the tumor size in immuno-deficient mice that were previously inoculated s.c. with human acute leukemia cells NB4 or MM cells RPMI8226 (p<0.01). Tumors from animals treated with T140 had smaller sizes and weights, larger necrotic areas and high apoptotic scores. Taken together, these data support the unique anti-cancer effect of T140 in hematological malignancies and indicate the potential therapeutic role of T140 in MM and leukemia patients. Disclosures: No relevant conflicts of interest to declare.

Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1372-1372
Author(s):  
Hendrik W. Van Deventer ◽  
Robert Mango ◽  
Jonathan Serody
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Mesenchymal Cells ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

The Xla (X-linked anemia) Mouse: A Transient Neonatal Anemia Caused by a Gata1 Splicing Mutation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3162-3162
Author(s):  
Kyle Miller ◽  
Michael Silvey ◽  
Derek Logsdon ◽  
Frederick Balch ◽  
Ndona Nsumu ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
X Chromosome ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Clonal Selection ◽  
Pcr Analysis ◽  
Nucleotide Change ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Erythroid Precursors

Abstract Abstract 3162 The Xla (X-linked anemia) mutant mouse was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and results in a severe and transient neonatal anemia. Xla/+ females exhibit severe anemia with 50% the level of red blood cell number, hematocrit and hemoglobin. Male Xla mice die in utero at 10.5 days gestation. The neonatal anemia observed in Xla/+ female pups is resolved by weaning age at 3 weeks by which time the mice present with a normal hematological phenotype. It is unknown how the neonatal anemia in Xla/+ females is alleviated. Previously, we mapped the Xla locus to the proximal end of the X chromosome near candidate gene Gata1 which showed no change in the coding sequence of GATA1 protein. Now we report the identification of a Gata1 mutation in Xla mice that results in an mRNA splicing defect. A nucleotide change (G to A) was identified 5 base pairs downstream of Exon 1E in intron 1 of the Xla Gata1 gene and results in the lack of incorporation of Exon 1E in the Gata1 mRNA expressed from the mutant locus. Therefore, in some erythroid lineage cells in Xla/+ mice, the normal 1E exon of Gata1 mRNA is replaced by Exon 1Eb/c which is known not to impact erythropoeisis since no GATA1 protein is made by this mRNA due to its inability to bind to ribosomes. These data show the Xla mouse results from a single nucleotide change impacting the normal splicing of the Gata1 gene. A second goal of this study was to understand why Xla/+ mice exhibit the neonatal transient anemia. A contributing factor is X chromosome inactivation which occurs in female mice during development. The short-term anemia in Xla mice was thought to be due to clonal selection of erythroid lineage cells characterized by the expression of GATA1 protein from the active X chromosome expressing only from the wild type Gata1 locus. Using an X-linked gene expressed in red blood cells (Pgk1, phosphoglycerate kinase 1) that varies between Xla mice and a wild derived strain, CAST/Ei, we examined the active state of the X chromosomes based on the expression of Pgk1 RNA in reticulocytes from hybrid Xla mice generated by breeding of these different strains. Examining expression of the X-linked Pgk1 SNP variant in the RNA of reticulocytes from hybrid Xla/+ mice reveals red blood cells are generated from two types of erythroid lineage cells. Pgk1 SNP RT-PCR analysis reveals that red blood cells not only derive from erythroid progenitors with the active X chromosome carrying the wild type Gata1 gene but also red blood cells are produced by erythroid lineage cells expressing the Xla mutant Gata1 mRNA on the active X chromosome (which does not make GATA1 protein). Therefore, some Xla erythroid cells derive from progenitors which express Gata1 transcripts using Exon 1Eb/c that does not stimulate erythropoiesis due to lack of GATA1 protein. The question is how these erythroid precursors generate normal red blood cells without the production of GATA1 protein. We hypothesize there is a developmentally expressed compensatory gene or pathway replacing GATA1 expression in GATA1-lacking erythroid precursors and required for the production of red blood cells in Xla mice. Analysis is underway to identify a potential novel gene or pathway impacting erythropoiesis in these mutant mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HFE Gene Mutation Status Predicts Response to Gemtuzumab Ozogamicin in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1307-1307 ◽  
Author(s):  
Etienne Paubelle ◽  
Alice Marceau ◽  
Florence Zylbersztejn ◽  
Mickael Dussiot ◽  
Ivan Cruz Moura ◽  
...  
Keyword(s):  
Total Dose ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Gemtuzumab Ozogamicin ◽  
Hfe Gene ◽  
Study Cohort ◽  
Dependent Manner ◽  
Wild Type ◽  
Significant Difference ◽  
Hfe Mutations

Abstract BACKROUND: The protein defective in type 1 hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins, associates with beta2-microglobulin and is implicated in membranous protein recycling. Gemtuzumab ozogamicin (GO), a monoclonal antibody directed against CD33 linked to a cytotoxic agent, has been used with controversial effects in acute myeloid leukemia (AML). Internalization of GO is required for its anti-leukemic effect. Therefore, we hypothesized that H63D or C282Y HFE gene mutations may impair GO activity by preventing its internalization. METHODS: Wild type, C282Y and H63D HFE leukemic cells and primary cell were used to assess effects of GO alone or in combination with cytarabine on cell proliferation and apoptosis. Flow cytometry, confocal and AMNIS stream analysis were used to evaluate GO internalization. HFE mutations were analyzed by PCR analysis on DNA from patients included in clinical studies. Post-hoc subgroup analysis was performed to assess in vivo the role of HFE status on GO efficacy and toxicity among the 280 patients of the ALFA-0701 study as a study cohort (patients aged 50-70 years; GO 3mg/m² on days 1, 4, and 7 of chemotherapy and on day 1 of the first and second induction ; total dose 15 mg/m² ) and then on the GOELAMS-LAM 2006 IR study (patients aged 18-60 years; GO 6 mg/m² on day 4 of chemotherapy during the induction and the first consolidation; total dose 12 mg/m²) and UK NCRI AML17 study (patients aged 18-81 years; GO 3 vs 6 mg/m² on day 1 of chemotherapy but not during consolidation) as validating cohorts. RESULTS: GO induced cell death by apoptosis in AML cell lines and primary cells in a dose-dependent manner and synergistically in combination with cytarabine. However, the IC5O of GO was significantly higher in HFE mutated cells (125 vs 10 ng/mL p<0.001). To further understand this phenomenon, the CD33 internalization was analyzed upon GO or anti-CD33 antibody exposure. In line with the cytotoxic effect, CD33 was significantly less internalized in HFE mutant cells (17.4% vs 65.1% after 1 hour, p<0.01). HFE mutations were screened in 242 of the 280 ALFA-0701 patients with DNA available. There were 155 non-mutated patients (64%), 68 (28%) heterozygous for H63D, 11 (5%) heterozygous for C282Y, and 8 (3%) homozygous for H63D, which is consistent with the prevalence of the various mutations in the French population. Median age was 62 years (50-71) and the M/F ratio was 0.5, equally distributed among the different groups. No significant difference was observed with respect to the diagnosis of various hematological parameters, including white blood cell count, blasts number, cytogenetic subgroups, molecular mutation incidences (FLT3, NPM1, CEBPA, IDH, DNMT3A). In the ALFA-0701 study cohort, HFE wild-type (WT) patients had a higher overall survival (OS) when treated in the GO arm (median, not reached vs 19.5 months, p=0.0193). In contrast, OS was similar among patients with heterozygotes HFE mutations treated in the GO and the control arm (median, 19.9 vs 21.9, p=0.9675). In confirmatory cohorts, GO treatment led to a trend to increased OS in the GOELAMS-LAM 2006 IR cohort only in HFE WT patients (4-year OS with GO 62% vs 48%, p=0.08) but not in mutated patients (4-year OS with GO 73% vs 64%, p=0.45). Furthermore, in this cohort in the FLT3 WT patients subgroup, GO further improve OS in WT patients (4-year OS with GO 72% vs 50%, p=0.017), but not in patients with heterozygous HFE mutation (4-year OS with GO 80% vs 65%, p=0.23) In the UK NCRI AML17 cohort, which used GO only during induction, 245 patients were randomized between GO at 3mg/m2 and 6mg/m2 and evaluated for HFE status. Overall there was no effect of GO dose on outcomes, and no evidence of either any heterogeneity by HFE, nor any subgroup, which showed a differential effect of GO dose. CONCLUSIONS: Future studies should focus on optimising the fractionated schedule for GO at the 3mg/m2. Fractionated high doses (3x3mg/m2) during induction and single dose during consolidation seems to be the best schedule. Most importantly, the effect of GO treatment differed between HFE WT and heterozygote mutated AML patients. GO only increased OS among HFE WT patients. This is likely related to impaired internalization of the CD33 target. Our data suggest that HFE status should be used as a companion test to predict outcome of AML treated with GO. Disclosures No relevant conflicts of interest to declare.

Unscheduled re-entry into the cell cycle induced by NGF precedes cell death in nascent retinal neurones

2000 ◽  
Vol 113 (7) ◽  
pp. 1139-1148 ◽  
Author(s):  
J.M. Frade
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Ganglion Cells ◽  
Neurotrophin Receptor ◽  
Dependent Manner ◽  
Apoptotic Effect ◽  
Induced Apoptosis

During their early postmitotic life, a proportion of the nascent retinal ganglion cells (RGCs) are induced to die as a result of the interaction of nerve growth factor (NGF) with the neurotrophin receptor p75. To analyse the mechanisms by which NGF promotes apoptosis, an in vitro culture system consisting of dissociated E5 retinal cells was established. In this system, NGF-induced apoptosis was only observed in the presence of insulin and neurotrophin-3, conditions that favour the birth of RGCs and other neurones expressing the glycoprotein G4. The pro-apoptotic effect of NGF on the G4-positive neurones was evident after 10 hours in vitro and was preceded by a significant upregulation of cyclin B2, but not cyclin D1, and the presence of mitotic nuclei in these cells. Brain-derived neurotrophic factor prevented both the increase of cyclin B2 expression in the G4-positive neurones and the NGF-induced cell death. Finally, pharmacologically blocking cell-cycle progression using the cyclin-dependent kinase inhibitor roscovitine prevented NGF-induced cell death in a dose-dependent manner. These results strongly suggest that the apoptotic signalling initiated by NGF requires a driving stimulus manifested by the neuronal birth and is preceded by the unscheduled re-entry of postmitotic neurones into the cell cycle.

Lysosomal Degradation of CD33, the Target for the Anti-Leukemia Immunoconjugate, Gemtuzumab Ozogamicin: Evidence for Cbl-Mediated Monoubiquitination.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2469-2469
Author(s):  
Roland B. Walter ◽  
Brian W. Raden ◽  
Darren M. Kamikura ◽  
Irwin D. Bernstein ◽  
Jonathan A. Cooper
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Lentiviral Vector ◽  
Gemtuzumab Ozogamicin ◽  
Jurkat Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Src Family Kinase ◽  
Lysosomal Degradation ◽  
Src Tyrosine Kinase

Abstract Background: CD33, the target for the anti-leukemia immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), is a transmembrane glycoprotein that contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Although we have previously shown that disruption of the ITIMs prevents effective uptake of antibody-bound CD33 and significantly reduces GO-induced cytotoxicity, the mechanisms underlying this uptake and intracellular trafficking of the antibody-CD33 complex are not known. In this study, we tested whether CD33 is a target for monoubiquitination, a posttranslational modification that marks proteins for lysosomal degradation, and how this modification relates to tyrosine phosphorylation and endocytosis. Methods: CD33− Jurkat and 32D cells transduced with a lentiviral vector expressing either wild-type or mutant CD33 were analyzed for CD33 expression as well as internalization of anti-CD33 antibody by flow cytometry. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33 or TKB domain of Cbl/Cbl-b, using human myeloid cell lysates. For co-immunoprecipitation experiments, constructs encoding wild-type and mutant CD33, ubiquitin, Cbl/Cbl-b, and wild-type Fyn were transfected into HEK293T cells. Results: In engineered Jurkat cells, treatment with either anti-CD33 antibody or the tyrosine phosphatase inhibitor pervandate increased tyrosine phosphorylation of CD33. Pervanadate enhanced uptake of antibody-bound CD33; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2. CD33 interacted with the TKB domains of Cbl and Cbl-b in GST fusion protein pulldown assays. Similarly, Cbl and Cbl-b were co-immunoprecipitated with CD33 in transfected 293T cells. Experiments in 293T cells further showed that CD33 is monoubiquitinated in an ITIM-dependent manner. Cbl or Cbl-b significantly increased the amount of CD33-associated ubiquitination; Cbl/Cbl-b-dependent ubiquitination could further be enhanced by co-transfected Fyn. Finally, 32D cells expressing lysine-to-arginine mutants of CD33 displayed much higher levels of surface CD33 but had reduced internalization of antibody-bound CD33 compared to cells expressing wild-type CD33, consistent with a reduced internalization/degradation of the lysine-to-arginine mutants. Conclusion: These studies indicate that Src-family kinase dependent phosphorylation favors internalization of antibody-bound CD33 and identify Cbl family proteins as potential binding partners of CD33. Importantly, CD33 is a target for ITIM-dependent monoubiquitination, and Cbl family proteins can act as an E3 ligase in this reaction. Our data therefore suggest a model where lysosomal routing and degradation of antibody-bound CD33 is secondary to Src-family kinase-induced phosphorylation of CD33 with subsequent phosphotyrosine-dependent binding of Cbl to and monoubiquitination of CD33.

Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1384-1384 ◽  
Author(s):  
Russell R. Hoover ◽  
Matthew W. Harding
Keyword(s):  
Clinical Trials ◽  
Cell Viability ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Leukemia Cell ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Dependent Manner ◽  
Wild Type

Abstract MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.

DYRK3 Encodes an Inhibitor of Erythroid Differentiation and Is Expressed at High Levels in Patients with Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3661-3661
Author(s):  
David P. Steensma ◽  
Julie C. Porcher ◽  
Antony Chadderton ◽  
Kevin Laubscher ◽  
Deb Jaworski ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Erythroid Differentiation ◽  
Murine Models ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Anemia Of Chronic Disease ◽  
Orally Bioavailable

Abstract Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis. Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor. Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied. Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.

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