The Effects of the Neutrophil Elastase Inhibitors MK0339 and Sivelestat on the Survival, Proliferation and Maturation of iPSC and HL60 Cells Expressing Mutant Neutrophil Elastase

Abstract Background : Mutations in ELANE, the gene for neutrophil elastase (NE), are the most frequent cause of both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). G-CSF and HSCT are currently the only effective treatment options. We have examined the possibility that inhibitors of NE might also be treatment options. Methods: Cells: HL60 human promyelocytic cells, a commercially available cell line, and induced pluripotent stem cells (iPSc) derived from patient dermal fibroblasts or bone marrow stromal cells, reprogrammed using episomal vectors. NE inhibitors: Two cell permeable inhibitors were used: MK0339 provided by Merck and sivelestat from Sigma. Results: Expression of mutant NE in HL60 Cells Transient Expression:Transient transfection of HL60 cells with 4 different ELANE mutants (P139L, C151Y, V174_C181del, G214R) cloned in pcDNA 3.1 vector showed increased apoptosis after 48h of culture in 10% FBS RPMI and expression of mutant constructs. Addition of Merck NE inhibitor MK0339 with 1uM final concentration in culture significantly improved survival of cells expressing each of the mutations tested detected by Annexin V staining using flow cytometry. (p<0.0001) The effect of the sivelestat (100nM) was significantly effective on only G214R mutant. (p= 0.035) Regulated Expression:We tested the effects of Merck inhibitor MK0339 on tet-inducible HL60 cells with regulated expression of mutant NE (V174_C181del), a mutation found in a patient with severe congenital neutropenia who died of AML. We examined 3 characteristics of these cells: cell survival, myeloid differentiation, and proliferation. The cell lines, expressing wild type or mutant NE under a tetracycline regulated promoter, were treated with MK0339 and cultured for 72 hours in the presence or absence of doxycycline. MK0339 significantly reduced mutant NE triggered apoptosis and increased granulocytic differentiation induced by addition of all-trans retinoic acid (ATRA) in 5-day cultures. Expression of mutant NE also significantly reduced the growth rate of the HL60 cells, but addition of MK0339 promoted growth of these HL60 cells. Induced Pluripotent Stem Cells (iPSC) We have established iPS cell lines from three neutropenic patients (two brothers with P139L and another patient with G214R) and two healthy volunteers. Cell lines from all 3 patients consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers. We therefore tested the effects of MK-0339 and sivelestat under the same culture conditions. Growth of patient cell lines was enhanced and the cells appeared more robust, especially after addition of 1uM MK0339. The latter normalized patient derived iPSc proliferation. We also investigated survival of the iPS cells during growth in feeder free settings and the effect of the NE inhibitors. After 4 days of culture in DEF-CS system, the patient derived cell lines showed increased apoptosis measured by Annexin V. We observed more than 9-fold difference in increased apoptosis of patient derived cells compared to healthy volunteers. Addition of MK-0339 and sivelestat reduced the apoptosis. MK0339 significantly reduced the apoptosis in both mutant cell lines, while sivelestat significantly reduced the apoptosis in only P139L mutant cell lines. Again, sivelestat appeared to be less effective. Myeloid differentiation of iPSc was also impaired and inefficient in patient derived cell lines detected with granulocytic differentiation markers using flow cytometry. Introduction of MK0339 in this system normalizes the impaired myeloid differentiation capacities of patient derived iPSc lines. Summary/Conclusion: These studies clearly show effects of these selected inhibitors to enhance neutrophil formation in reproducible cellular models that mimics features of SCN and CyN. We believe this work will lead to novel therapies for ELANE associated neutropenia and open the door to other new therapies for neutropenia. Disclosures Dale: Amgen: Consultancy, Honoraria, Research Funding.

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CRISPR/Cas9 Knock-in HL60 Cells Closely Simulate Cellular and Functional Abnormalities of ELANE associated Neutropenia; Phenotype Rescue with MK-0339 Neutrophil Elastase Inhibitor

Blood ◽

10.1182/blood-2018-99-113054 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3683-3683

Author(s):

Vahagn Makaryan ◽

Breanna Fletcher ◽

Merideth L. Kelley ◽

Masoud Nasri ◽

Julia Skokowa ◽

...

Keyword(s):

Cell Lines ◽

Neutrophil Elastase ◽

Mutant Cell ◽

Annexin V ◽

Hl60 Cell ◽

Myeloid Differentiation ◽

Wild Type ◽

Granulocytic Differentiation ◽

Congenital Neutropenia ◽

Hl60 Cells

Abstract Background: Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. Congenital neutropenia is characterized by low neutrophil counts in peripheral blood and impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. To date, more than 100 different ELANE mutations have been reported. We previously described that some mutations (e.g. G214R and C151Y) are associated with more severe outcomes, while others (e.g. P139L and R220Q) result in relatively mild clinical phenotypes. (Curr Op Hematol. 2015;22:3-11) To explain the biological effects of various ELANE mutants we have created CRISPR/Cas9 edited ELANE mutants using the commercially available human promyelocytic cell line HL60. Methods: We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with ELANE P139L and C151Y single point heterozygous mutations. Both ELANE mutant cell lines as well as wild-type HL60 cells were cultured for 7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. Unfolded protein response (UPR) was measured by western blotting using UPR specific antibodies. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have recently reported that MK-0339 increases cell survival and myeloid differentiation in cellular models of ELANE associated neutropenia. (Makaryan, et al, J Leukoc Biol. 2017;102(4):1143-1151). We examined the effects of MK-0339 on these cell lines. Results: Annexin V staining showed more than 2-fold increase in apoptotic cells in both mutant cell lines compared to wild-type. Granulocytic differentiation measured by surface CD11b expression was significantly impaired in both mutant cell lines (p-values <0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature myelocytes. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. It is important to note that the C151Y mutant, the mutant clinically associated with more severe disease, shows more severe impairment compared to P139L. Addition of 1uM MK-0339 to the culture completely restored normal survival and myeloid differentiation of both mutant cell lines. Conclusions: We believe CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating genetic neutropenias. These results suggest that a panel of different mutant ELANE HL60 cell lines will help to elucidate the molecular and biochemical origin of phenotypic variability in ELANE associated neutropenia. Disclosures Dale: Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy.

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CRISPR/Cas9 Mediated ELANE Knock-out Restores Survival and Granulocytic Differentiation of HL60 Cells Expressing Mutant Neutrophil Elastase: Is Neutrophil Elastase a Dispensible Granulocyte Protease?

Blood ◽

10.1182/blood-2019-124988 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 435-435

Author(s):

Vahagn Makaryan ◽

Isabella N. Archibald ◽

Merideth L. Kelley ◽

Breanna Fletcher ◽

David C. Dale

Keyword(s):

Cell Line ◽

Cell Lines ◽

Neutrophil Elastase ◽

Mutant Cell ◽

Annexin V ◽

Hl60 Cell ◽

Myeloid Differentiation ◽

Granulocytic Differentiation ◽

Congenital Neutropenia ◽

Knock Out

Background : Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. More than 130 different ELANE mutations have been found in patients with cyclic and congenital neutropenia, and genotype-phenotype studies suggest that specific mutations cause more severe disease. (Curr Op Hematol. 2015;22:3-11) Mutant NE is also implicated as the primary cause of ELANE associated neutropenia by studies showing that cell permeable inhibitors of NE correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). Hypothesis: If inhibition of NE can correct the cellular defect, deletion of ELANE will have similar effects and permit studies to see if NE is an essential neutrophil protease. Methods: We used CRISPR/Cas9 mediated gene editing to create a cellular model of congenital neutropenia in the commercially available human promyelocytic cell line, HL60. We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with knock-in (KI) ELANE P139L, C151Y and G214R single point heterozygous mutations. Using the same technology, we then performed ELANE gene complete knock out (KO) of ELANE in each mutant cell line. All engineered cell lines as well as wild-type HL60 cells were cultured for 5-7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. We measured the unfolded protein response (UPR) by western blotting using UPR specific antibodies, phagocytosis with E. coli particles using fluorescence detection, IL-8 stimulated chemotaxis in trans-well system and PMA activated respiratory burst by flow cytometry. Results: Cells expressing the P139L and C151Y mutant ELANE had increased Annexin V staining more than a 2-fold increase in apoptotic cells at 7 days in culture. Granulocytic differentiation, measured by surface CD11b expression, was significantly impaired. (p<0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature neutrophils in 7-day cultures. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. Chemotaxis to the IL8 chemo-attractant was severely impaired in cells expressing mutant NE. Respiratory burst and phagocytosis was also altered in cells expressing mutant NE. ELANE KO corrected all these cellular and functional abnormalities, reverting these functions toward the wild phenotype. Studies are in progress for the third cell line expressing the G214R mutation. Conclusions: CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating ELANE associated neutropenia. The presence of mutant NE severely impairs neutrophil functions, and its deletion through KO of ELANE corrects the cellular abnormalities and reverses the induced abnormalities. These results suggest that CRISPR/Cas9 mediated genetic knock down of NE is a novel therapeutic approach for treatment of this disorder. This data also suggests that total absence of NE does not alter basic functions of granulocytes. Disclosures Dale: Sanofi Aventis: Consultancy, Honoraria; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.

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Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽

10.1182/blood.v116.21.2474.2474 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2474-2474

Author(s):

Jesus Duque-Afonso ◽

Aitomi Essig ◽

Leticia M Solari ◽

Tobias Berg ◽

Heike L. Pahl ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Functional Role ◽

Target Gene ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Leukemia Cells ◽

Myeloid Differentiation ◽

Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

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Myelopoiesis From Induced Pluripotent Stem Cells Reveals The Role Of Elastase Activity In The Pathogenesis Of Severe Congenital Neutropenia

Blood ◽

10.1182/blood.v122.21.442.442 ◽

2013 ◽

Vol 122 (21) ◽

pp. 442-442

Author(s):

Ramesh C Nayak ◽

Lisa Trump ◽

H. Leighton Grimes ◽

Carolyn Lutzko ◽

Jose A. Cancelas

Keyword(s):

Stem Cell ◽

Neutrophil Elastase ◽

Embryoid Body ◽

Point Mutations ◽

Annexin V ◽

High Dose ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Elastase Activity ◽

Induced Pluripotent

Severe congenital neutropenia (SCN) is a hereditary neutropenia characterized by granulocytic precursor differentiation arrest in which the pathogenic mechanism is poorly understood. Over half of SCN patients associate with autosomal dominant point mutations in the gene encoding neutrophil elastase (ELANE). High-dose G-CSF therapy succeeds in increasing neutrophil counts in many SCN patients, but the molecular explanation for high dose G-CSF rescue is unknown. Mutations in the G-CSF receptor (CSF3R) associated with a dramatic risk for transformation provides a cautionary note for high dose G-CSF treatment. The pathogenic mechanism of ELANE mutations is incompletely understood due to the lack of disease recapitulation in murine models of ELANE-mutant hematopoiesis and the difficulty to obtain primary, relevant myeloid cell populations from patients. Using induced pluripotent stem cell (iPSC) derived myelopoietic cultures from peripheral blood mononuclear cells of patients with ELANE exon 3 point mutations (Q97P and I118N), we designed experiments to recapitulate G-CSF signaling in SCN myelopoiesis. The patient-derived iPSC lines were characteristically similar to human embryonic stem cell lines and normal blood-derived iPSC lines with expression of SSEA-4, Tra-1-60, Tra-1-81, and CD9 pluripotency markers >85% in all lines as determined by FACS analysis at passage 10-15. All iPSC retained a normal karyotype and ELANE locus mutations of the original specimens. Both SCN iPSC lines and control iPSC lines (normal and immune-mediated congenital neutropenia) show functional G-CSF (50 ng/mL)-induced differentiation into the myeloid lineage. However, G-CSF failed to induce terminal granulocytic differentiation of SCN iPSC derived myeloid progenitors resulting in differentiation arrest at the promyelocyte/myelocyte stage. The hematopoietic cells derived from SCN iPSC line show reduced frequency of CFU-G and CFU-GM (CFU-G+CFU-GM, 22±1.15 in 104 embryoid body cells from non SCN iPSC vs 4±2 in 104 embryoid body cells from ELANE mutated SCN iPSC). Moreover, SCN iPSC promyelocytes show significantly increased apoptosis over controls as determined by Annexin-V analysis (31% Annexin-V+ in SCN iPSC derived promyelocyte vs 3% in control iPSC). Surviving SCN promyelocytes showed decreased reactive oxygen species activity and no phagocytic activity. In contrast, suprapharmacological doses of G-CSF (1000 ng/mL) rescued SCN iPSC-derived differentiation. It has been shown that NE, expressed in promyelocytes, can cleave recombinant G-CSF and CSF3R. We evaluated the possibility that inadequate G-CSF signaling in SCN iPSC was due to abnormal elastase activity. To this end, we supplemented 50 ng/mL G-CSF cultures with a specific, cell-permeant neutrophil elastase inhibitor (Sivelastat, 230 nM) to find rescued granulocyte differentiation arrest (31% neutrophil in myeloid differentiation culture of SCN iPSC derived hematopoietic progenitors in presence of Sivelastat vs 2% mature neutrophils without Sivelastat). In conclusion, SCN modeling through patient iPSC derived myelopoiesis and G-CSF driven granulocytic recapitulation unveil a mechanism of resistance to G-CSF therapy with important translational implications for therapy. Combination of low-dose G-CSF and NE inhibitory therapy may result in an efficacious, safer approach in SCN therapy. Disclosures: No relevant conflicts of interest to declare.

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KMT2E-Mediated Epigenetic Reprogramming Promotes the Sensitivity to All-Trans Retinoic Acid and Increases the Granulocytic Differentiation in AML Cells

Blood ◽

10.1182/blood-2018-99-118478 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3838-3838

Author(s):

Luciana Yamamoto Almeida ◽

Isabel Weinhäuser ◽

Diego Antonio Pereira-Martins ◽

Josiane Lilian Schiavinato ◽

Patrícia Vianna Bonini Palma ◽

...

Keyword(s):

Cell Lines ◽

Tumor Volume ◽

Tumor Burden ◽

Apoptotic Index ◽

Myeloid Differentiation ◽

Dependent Manner ◽

Granulocytic Differentiation ◽

Nsg Mice ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid

Abstract Background: The histone-lysine N-methyltransferase 2E (KMT2E) is a member of the KMT2 family that regulates terminal myeloid differentiation and hematopoietic stem cell self-renewal. However, unlike the other members of KMT2 family, KMT2E does not exhibit the intrinsic histone methyl transferase activity, but has been shown to indirectly interact with chromatin by regulating SET7/9 expression and directing the tri-methylation state of the histone H3K4 (H3K4me3). We previously reported that the low expression of KMT2E predicts poorer prognosis in acute promyelocytic leukemia (APL) and that the overexpression of KMT2E contributed to granulocytic differentiation of APL cells in response to all-trans retinoic acid (ATRA). ATRA as a single agent is not effective in cell differentiation of non-APL acute myeloid leukemia (AML) but changes in H3K4 methylation promotes ATRA sensitivity and upregulates myeloid-differentiation-associated genes in AML cells. Therefore, to better understand the role of KMT2E in leukemogenesis and to test whether ATRA was capable to epigenetically mediate the KMT2E function in non-APL AML blasts, we investigated the effect of KMT2E overexpression in the cell viability, tumor growth and signaling response to ATRA in vitro and in vivo. Methods: qPCR analyzes revealed that the AML cell lines OCI-AML3, U937 and THP-1 express low levels of KMT2E in relation to the APL cell line NB4 and then, they were selected for further experiments. OCIAML3, THP1, and U937 were transduced with the pMEG or the pMEG-KMT2E vector, and U937 cells were also submitted to KMT2E gene silencing by a specific shRNA. The transduced cells were subjected to ATRA (1µM) treatment to evaluate proliferation (Ki-67), clonogenicity, cell cycle and apoptosis (annexin V). For dose-response curves (MTT), cells were administrated graduated ATRA concentrations and the IC50 values were calculated. ATRA-induced granulocytic differentiation was determined by immunophenotyping analysis of myeloid markers: CD11b, CD11c, CD14, CD16, CD64, and HLA-DR. Genes (SETD7/9) and protein (H3K4me3) expressions were established by qPCR and Western blot analysis respectively. 8-10 week-old NSG mice received a dorsal s.c. injection of 5 x 106 U937 cells (transduced with empty/KMT2E/shKMT2E) and 7 days after initial tumor growth treatment was initiated by intraperitoneal injection of the vehicle (n=26) or ATRA (5 mg/kg, n=30) for another 7 days. Tumor volume (V) was obtained using the formula (V=W2 x L x 0.52), where W represents the smaller and L the large diameter. The tumor apoptotic index was determined by TUNEL assay. Results: KMT2E overexpression significantly increased cell proliferation and the proportion of G2/M cells in all cell lines (all P<.01) but no differences in clonogenicity were observed. The IC50 value of ATRA for U937 was of 23 µM for cells transduced with empty vector and 11 µM for pMEG-KMT2E cells, while for THP1 empty/KMT2E cells received a dose of 9.93 µM and 1.68 µM respectively. ATRA increased apoptosis in a time-dependent manner in U937 and THP1 cells (all P<.05), but not in OCIAML3 p-MEG-KMT2E cells. Furthermore, ATRA-treated U937 and THP1 cells presented G0/G1-cell cycle arrest (all P<.05) after 72 hours, while no effect was observed in OCI-AML3. ATRA-induced granulocytic differentiation increased the CD11b/CD14 expression in U937 and THP1-KMT2E cells, within 72 hours of treatment compared to controls (all P<.05). ATRA treatment enhanced expression of H3K4me3 in a time-dependent manner which was correlated with enhanced transcript levels of SETD7/9 (all P<.05). In NSG mice, ATRA significantly reduced the tumor burden in U937- KMT2E cells and increased the apoptotic index (P<.01) after 7 days of treatment, albeit the high tumor burden in KMT2E cells treated with the vehicle. As a result the mean±SD of tumor volume for empty/KMT2E cells was 1296±95.6 mm3 and 445±59.6 mm3, respectively (P=.001). Finally, U937 shKMT2E indicated no difference in ATRA sensitivity and tumor volume (840±54.8 mm3 and 550±32.1 mm3 for empty/shKMT2E cells, respectively (P>.05). Conclusion: Our results demonstrated that KMT2E increases the cellular proliferation and ATRA-induced granulocytic differentiation in non-APL AML cell lines. Furthermore, ATRA triggered epigenetic reprogramming resulting in increased levels of H3K4me3, which redirected the biological function of KMT2E. Disclosures No relevant conflicts of interest to declare.

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Src Kinase Inhibitors Enhance ATRA-Mediated Differentiation of Myeloid Cells.

Blood ◽

10.1182/blood.v108.11.4264.4264 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4264-4264

Author(s):

Michelle B. Miranda ◽

Daniel E. Johnson

Keyword(s):

Retinoic Acid ◽

Cell Lines ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Src Family Kinases ◽

Myeloid Differentiation ◽

Monocytic Differentiation ◽

Granulocytic Differentiation ◽

Current Standard ◽

The Impact

Abstract Myeloid leukemias are characterized by a blockade in differentiation resulting in accumulation of proliferating progenitor cells and a lack of mature granulocytes and monocytes. In acute promyelocytic leukemia (APL), a subtype of AML, treatment with all-trans retinoic acid (ATRA) has been shown to overcome this blockade in differentiation and is the current standard of care for APL patients. However, other forms of myeloid leukemia show no response to ATRA. Src family kinases (SFK) have been shown to be activated during normal myeloid development by both proliferation-inducing cytokines, such as IL-3, and differentiation-promoting factors, such as G-CSF. To elucidate the role of src family kinases during myeloid differentiation, we examined the impact of SFK inhibitors, PP1 and PP2 on ATRA-mediated differentiation of myeloid cell lines. Interestingly, PP1 potentiated ATRA-mediated myeloid differentiation of HL-60 cells, inducing a two-fold increase in differentiated cells at 72 hours as determined by CD11b staining, NBT reduction and morphological analysis. To determine whether enhancement of ATRA-mediated differentiation was specific for PP1, or could be promoted by a different src family kinase inhibitor, we evaluated the impact of PP2. PP2 was found to exhibit similar effects on ATRA-induced myeloid differentiation of HL-60 cells. Additionally, both PP1 and PP2 enhanced ATRA-induced monocytic differentiation of U937 cells and granulocytic differentiation of NB-4 cells. Interestingly, PP1 was found to enhance ATRA-induced differentiation even when added 24 hours after addition of ATRA. The impact of PP1 on ATRA-mediated myeloid differentiation was dependent on signaling via the MEK/ERK pathway since pre-treatment with U0126, a MEK-1/-2 inhibitor, attenuated myeloid differentiation induced by the combination of RA and PP1. Reporter assays utilizing an RARE-luciferase construct indicate that PP1 and PP2 do not enhance ATRA-mediated differentiation by promoting the transcriptional activity of the retinoic acid receptor (RAR). Together, our results demonstrate that PP1 and PP2 potently enhance ATRA-induced myeloid differentiation of AML cell lines and suggest that src inhibition in combination with ATRA may be a useful treatment for AML. Studies evaluating the impact of src inhibition on ATRA-mediated differentiation of primary AML cells are currently underway.

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Vitamin D3 and Lenalidomide Synergize To Induce Apoptosis In Mantle Cell Lymphoma Via Demethylation Of BIK

Blood ◽

10.1182/blood.v122.21.1830.1830 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1830-1830

Author(s):

Carole Brosseau ◽

Christelle Dousset ◽

Cyrille Touzeau ◽

Sophie Maiga ◽

Philippe Moreau ◽

...

Keyword(s):

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Vitamin D3 ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Treatment Options ◽

Annexin V ◽

Dna Demethylation ◽

Mantle Cell

Abstract Among new treatment options for mantle cell lymphoma (MCL), the targeted drug lenalidomide appears as one of the most efficient molecules. Lenalidomide has multiple modes of action targeting the tumor cell and its environment including the immune system. It is widely reported that cancer patients are deficient in vitamin D3 (1,25-dihydroxyvitamin D3, VD3) and recent studies have shown in non Hodgkin lymphomas (NHL) that VD3 levels have a prognostic value on survival (Drake, J Clin Oncol. 2010;28:4191). While the relations between VD3 and cancer incidence remain unresolved, it has been shown that VD3 displays anti-tumoral properties via its anti-proliferative, pro-differentiation, anti-inflammatory and anti-angiogenic properties. We assessed the efficacy of VD3 to potentiate cell death induced by lenalidomide in MCL cell lines and patients’ samples and explored the mechanisms of cell death in this context. Experiments were conducted on a panel of 6 MCL cell lines (JEKO-1, MINO, GRANTA-519, UPN-1, REC-1 and Z138) and 8 primary peripheral blood samples. After 6 days of treatment, MCL cells were weakly sensitive to low doses of lenalidomide (1µM and 10µM for cell lines and samples, respectively). Addition of physiological doses of VD3 (100nM) significantly and synergistically increased cell death in 67% of cell lines (Z138, JEKO-1, MINO, REC-1) and in 63% of primary samples (p<0.05). However resistance to lenalidomide alone was not reversed by VD3 since both GRANTA-519 and UPN-1 remained unsensitive. Apoptosis, characterized by Annexin V staining, appearance of a subG1 peak and caspase 9 activation, was dependent on Bax expression, since transient extinction of BAX by siRNA in JEKO-1 cells inhibited cell death (mean of inhibition 30%±5%, p=0.03). The combination of lenalidomide and VD3 dramatically increased expression of the BH3-only Bik (Bcl2-Interacting Killer) protein in sensitive (Z138, JEKO-1, MINO, REC-1) but not resistant (GRANTA-519, UPN-1) cell lines, without affecting the expression of other molecules of the Bcl2 family. By immunoprecipitation assays, we showed that induced-Bik was not bound to the anti-apoptotic molecules Bcl2, BclxL or Mcl1 in treated cells but was free to activate such pro-apoptotic molecules as Bax. Moreover, siBIK RNA significantly decreased the proportion of Annexin V+ cells observed after treatment with lenalidomide and VD3, respectively by 36%±9% (p=0.04) and 28%±4% (p=0.04) in JEKO-1 and MINO cells. This confirmed the involvement of Bik in the cell death induced by this synergistic combination. Q-RT-PCR assays disclosed that Bik accumulation was related to an increase in BIK mRNA expression. BIK expression is controlled by the transcription factor TEF and is regulated by epigenetic modifications, its expression being silenced by methylation in many cancer cells. We showed that Bik accumulation induced by lenalidomide and VD3 was not related to an increase in TEF expression. To determine whether Bik expression could be induced or increased upon demethylation in MCL, we treated cell lines for 3 days with 1µM 5-azadecytidine (5-aza). Indeed, higher expression of Bik was observed after this treatment in the four cell lines sensitive to lenalidomide. Of note, cell death induced by 5-aza correlated linearly to that induced by lenalidomide and VD3 (p<0.001, r=0.95, n=6), suggesting that BIK demethylation could be a key point in the response to this combination. To directly assess the level of BIK methylation in MCL cell lines, we then performed a DNA methylation specific PCR assay on bisulfite-treated DNA, which targets the CpG rich region located within intron 1, as previously described by Hatzimichael et al (Leuk Lymphoma. 2012;53:1709). Indeed, we showed that lenalidomide and VD3 increased the proportion of unmethylated over methylated BIKDNA CpG islands in sensitive (2 to 5-fold increase) but not in resistant cell lines. These data show that the association of lenalidomide and VD3, by increasing BIK expression through DNA demethylation, is an efficient combination to induce the apoptosis of MCL cells. They also underline the interest of measuring the level of VD3 in MCL patients especially those receiving lenalidomide, since supplementation in deprived patients might improve the effect of therapy. Disclosures: No relevant conflicts of interest to declare.

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A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation

Blood ◽

10.1182/blood-2013-06-508887 ◽

2014 ◽

Vol 123 (8) ◽

pp. 1239-1249 ◽

Cited By ~ 29

Author(s):

Olga Klimenkova ◽

Wienke Ellerbeck ◽

Maksim Klimiankou ◽

Murat Ünalan ◽

Siarhei Kandabarau ◽

...

Keyword(s):

Protease Inhibitor ◽

Neutrophil Elastase ◽

Secretory Leukocyte Protease Inhibitor ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Granulocytic Differentiation ◽

Congenital Neutropenia ◽

Key Points ◽

Natural Inhibitor

Key Points The natural inhibitor of neutrophil elastase, SLPI, is severely reduced in severe congenital neutropenia patients. SLPI controls myeloid differentiation by regulation of NFκB, ERK1/2:LEF-1, and c-myc activation.

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The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

Blood ◽

10.1182/blood-2008-07-170282 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3299-3308 ◽

Cited By ~ 46

Author(s):

Vivian G. Oehler ◽

Katherine A. Guthrie ◽

Carrie L. Cummings ◽

Kathleen Sabo ◽

Brent L. Wood ◽

...

Keyword(s):

Retinoic Acid ◽

Tyrosine Kinase ◽

Cell Lines ◽

Kinase Inhibitor ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Chronic Phase ◽

Myeloid Differentiation ◽

Tyrosine Kinase Domain ◽

Granulocytic Differentiation

Abstract The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

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Effects of anti-CD44 monoclonal antibodies on differentiation and apoptosis of human myeloid leukemia cell lines

Blood ◽

10.1182/blood.v99.1.290 ◽

2002 ◽

Vol 99 (1) ◽

pp. 290-299 ◽

Cited By ~ 78

Author(s):

Rachida-Sihem Charrad ◽

Zeineb Gadhoum ◽

Junyuan Qi ◽

Anne Glachant ◽

Michèle Allouche ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cell Lines ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Terminal Differentiation ◽

Myeloid Differentiation ◽

Hl60 Cells ◽

Cellular Models

Acute myeloid leukemia (AML) is a heterogeneous leukemia characterized by the blockage of myeloid differentiation at different stages, which define distinct AML subtypes. We have recently reported that the ligation of CD44 with 2 activating monoclonal antibodies (mAbs), A3D8 and H90, triggers terminal differentiation of leukemic blasts in AML-M1/2 to AML-M5 subtypes, which are the most frequent ones. However, fresh AML blasts have short in vitro lifespans. Therefore, to find relevant in vitro cellular models for further studying the mechanisms involved in CD44-induced differentiation, we investigated whether CD44 ligation with A3D8 and H90 mAbs can induce terminal differentiation of THP-1, NB4, and HL60 cells, each interesting models of AML-M5 (monoblastic subtype), AML-M3 (promyelocytic subtype), and AML-M2 (myeloblastic subtype), respectively. We also study whether CD44 ligation induces a loss of proliferative capacity, an important feature of late-stage myeloid differentiation. In the second part of our study, we investigated whether A3D8 and H90 anti-CD44 mAbs can induce the differentiation and inhibit the proliferation of KG1a cells, which are very immature AML-M0 blasts. Using functional, antigenic, and cytologic criteria, we presently show that A3D8 and/or H90 induce terminal differentiation of THP-1, HL60, and NB4 cell lines and strongly inhibit their proliferation. Interestingly, cell-specific effects of H90 and A3D8 are observed. We also observe that incubation with A3D8 for 3 to 6 days induces an apoptotic cell death that is moderate in the case of THP-1 and HL60 cells and massive in the case of NB4 cells. Finally, our results demonstrate for the first time that it is possible to reverse the leukemic blockage of KG1a cells by using both an anti-CD44 mAb and retinoic acid. This result may provide a new experimental basis for a differentiative therapy in AML-M0 patients.

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