VASP Protein, a Binding Partner Of BCR-ABL and FAK, May Be Implicated In The Chronic Myeloid Leukemia Phenotype

Abstract Background VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP is a substrate of BCR-ABL oncoprotein and is tyrosine-phosphorylated in leukemic cells. However, the function of VASP and Zyxin in hematopoietic cells, in the BCR-ABL pathway and its participation in chronic myeloid leukemia (CML) remains unknown. Aims To analyze VASP and Zyxin expression in bone marrow cells from CML patients and healthy donors, as well the involvement of these proteins in hematopoietic cell differentiation and in the BCR-ABL signaling pathway. Materials and Methods VASP and Zyxin expression and phosphorylation were studied in bone marrow samples from 29 individuals (5 healthy donors, 5 CML patients at diagnosis, 16 CML patients responsive to treatment with tyrosine kinase inhibitors (ITK) and 3 CML patients resistant to ITK). One patient was analyzed at diagnosis and after ITK response. VASP or Zyxin silencing was performed by shRNA-lentiviral delivery in K562 cell line, an appropriated shControl was used. ShControl, shVASP and shZyxin K562 cells were induced to megakaryocytic differentiation with 20nM of PMA (phorbol myristate -13 -12 acetate) during 4 days and CD61 expression, a marker for maturing megakaryocytes, was verified by flow cytometry. During megakaryocytic differentiation, VASP and Zyxin gene expressions were evaluated by quantitative PCR; protein expression and activation were determined by Western blotting. Effector proteins of proliferation, apoptosis and adhesion in the BCR-ABL signaling pathway were analyzed in cells silenced for VASP or Zyxin. The interaction of VASP and BCR-ABL or FAK was evaluated by co-immunoprecipiation. Results Healthy donors showed p-VASP ser157 expression, in contrast to CML patients at diagnosis who did not present phospho-VASP ser157. After Imatinib treatment CML patients restored VASP phosphorylation however resistant patients maintained this absence. Zyxin showed the same expression in patients and healthy donors. During Imatinib treatment of K562 cells, phospho-VASP ser157 expression was increased and its interaction with BCR-ABL protein was reduced. VASP and Zyxin gene expressions were upregulated during megakaryocyte differentiation of K562 cells (8.7-fold increase, P=0.0115, and 3.6-fold increase, P=0.015, respectively). VASP and Zyxin protein expressions were increased during megakaryocytic differentiation, including the active form of these proteins (p-VASP ser157 and p-Zyxin ser142). VASP silencing in K562 cells resulted in a 40% decrease of CD61 expression at the end of the megakaryocytic differentiation (P<0.05). In addition, VASP and Zyxin silencing resulted in a decrease of BCL-2 and BCL-XL proteins. VASP binds to FAK, an adhesion effector protein of the BCR-ABL pathway, and it´s silencing resulted in a decreased phosphorylation of FAK y925. Conclusions In BCR-ABL cells, VASP and Zyxin modulated anti-apoptotic proteins and megakaryocytic differentiation. Hence, the altered expression of VASP activity in CML patients may contribute to the pathogenesis of the disease, affecting cellular differentiation or leukemic cell adhesion. Disclosures: No relevant conflicts of interest to declare.

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Bcr-Abl-Mediated Suppression of Normal Hematopoiesis in Chronic Myeloid Leukemia: Involvement of a Modified Form of NGAL.

Blood ◽

10.1182/blood.v106.11.2869.2869 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2869-2869

Author(s):

Hui Lin ◽

Xiaohong Leng ◽

Tong Sun ◽

Giuseppe Monaco ◽

Clifton Stephens ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

K562 Cells ◽

Soft Agar ◽

Leukemia Cells ◽

Active Process ◽

Culture Studies ◽

Induced Apoptosis

Abstract The BCR-ABL oncogene plays an essential role in chronic myeloid leukemia (CML). In NOD/scid mice injected with soft agar clones of a human CML cell line (K562), we observed a leukemia syndrome involving not only leukemia but also a severe reduction of normal mouse hematopoiesis (Lin et al., Oncogene, 2001). Some of these mice died of a wasting syndrome that involved suppression of hematopoiesis without extensive tumor cell invasion of the spleen and marrow. In CML patients, since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we postulate that this is an active process mediated by the leukemia cells. The lipocalin 24p3 is secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells (Devireddy et al., Science, 2001). We found that BCR-ABL+ mouse hematopoietic cells induce a persistent secretion of a modified form of 24p3 (21 kDa). Co-culture studies show that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Importantly, BCR-ABL+ hematopoietic cells are resistant to apoptosis under the same conditions. Conditioned medium (CM) from BCR-ABL+ cells expressing anti-sense/siRNA 24p3 or CM mixed with 24p3 antibody have reduced apoptotic activity for target cells. We also found that the expression of the Bcr-Abl oncoprotein and its tyrosine kinase are required for induction of 24p3 expression. Leukemic mice induced by BCR-ABL+ cells expressing anti-sense/siRNA 24p3 have increased levels of normal hematopoiesis (marrow and spleen erythropoiesis and blood platelet levels) and reduced invasion of leukemia cells in marrow and spleen tissues, but the leukemia cells readily invade liver and the abdomen as ascites (Lin et al, Oncogene, 2005). These findings indicate that suppression of normal hematopoiesis in BCR-ABL induced leukemia is an active process involving the apoptotic factor 24p3, raising the possibility that similar factors are involved in BCR-ABL+ CML patients. We have found that the K562 clones (Lin et al. 2001) have enhanced expression of NGAL (neutrophil gelatinase-associated lipocalin, human homologue of 24p3) transcripts compared to uncloned K562 cells. We generated additional soft agar K562 clones, each with different expression levels of NGAL transcripts. NOD/scid mice injected with the clone (C5) of K562 cell line expressing a high level of NGAL had severe depression of hematopoiesis and significantly shorter survival time as compared with mice injected with parental K562 cells and a clone (C6) expressing a low level of NGAL. Co-culture studies showed that the C5 K562 clone also induced apoptosis in BCR-ABL negative cells. We detected two glycosylated forms of NGAL/24p3 migrating at 24 kDa and 21 kDa on SDS-PAGE. The 21 kDa form is the major form in CM from mouse BCR-ABL+ cells and K562 clones. Our preliminary data with CML patient samples showed that levels of 21 kDa NGAL protein in bone marrow fluid correlated with BCR-ABL/ABL ratio. Further studies with more patient samples are ongoing to confirm the role of NGAL in suppressing normal hematopoiesis in CML patients and to determine the structural change(s) that leads to the modified form of 24p3/NGAL secreted by CML cells.

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The Cytoskeleton Proteins VASP and Zyxin Participate in Hematopoiesis and in the BCR-ABL Signaling Pathway.

Blood ◽

10.1182/blood.v120.21.2780.2780 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2780-2780

Author(s):

Vanessa Aline Bernusso ◽

Joao Machado-Neto ◽

Sara T. Olalla Saad ◽

Karin Spat Albino Barcellos

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

Cell Adhesion ◽

Cell Line ◽

Signaling Pathway ◽

K562 Cells ◽

Fold Increase ◽

Leukemic Cells ◽

Imatinib Treatment ◽

Megakaryocyte Differentiation

Abstract Abstract 2780 Background: VASP and Zyxin are cytoskeleton regulatory proteins. They act as a protein complex involved in the signal transduction for actin polymerization, in the control of cell adhesion, cell division and cellular motility. VASP and Zyxin are abnormally expressed in epithelial tumors and are related with tumor progress. VASP is a substrate of the BCR-ABL oncoprotein and is tyrosine-phosphorylated in BCR-ABL leukemic cells. However, the function of VASP and Zyxin in hematopoietic cells and in the BCR-ABL pathway is not yet known; in addition their possible participation in chronic myeloid leukemia (CML) remains an interesting issue to be clarified. Aims: To evaluate the effects of VASP and Zyxin silencing in cell proliferation, apoptosis and differentiation of BCR-ABL K562 cells. Methods: shRNA-lentiviral delivery was used to silence VASP and Zyxin expression in K562 cell line. The shRNA-lentiviral control, VASP and Zyxin cells were treated with different Imatinib concentrations (0, 0.1, 0.5 and 1μM) during 48 hours. Cellular proliferation was measured by MTT assay and apoptosis by flow cytometry with annexin-V. To differentiate cells into megakaryocytes, K562 cells were treated with 20nM of PMA during 4 days and cells were evaluated by the presence of CD61 and CD41 cell markers by flow cytometry. The expression of VASP and Zyxin in cells submitted to megakaryocyte differentiation was evaluated by quantitative PCR and western blotting; protein phosphorylation was also analyzed by western blotting. The interaction of BCR-ABL and VASP after imatinib treatment was evaluated by co-immunoprecipiation. Results: Zyxin silenced cells treated with 0.5μM and 1μM of Imatinib showed a decrease of 17% (P<0.05) and of 22% (P<0.01) in cell proliferation, respectively, compared to the control treated cells. In K562 cells treated with 1μM of Imatinib, VASP and Zyxin silencing increased apoptosis in 21% (P<0.05) and 40% (P<0.05), respectively. VASP and Zyxin gene expressions were upregulated during megakaryocyte differentiation of K562 cells (8.7-fold increase, P=0.0115, and 3.6-fold increase, P=0.015, respectively). In HEL cells (BCR-ABL negative cell line) VASP and Zyxin protein expressions were increased during megakaryocyte differentiation, including the active form of these proteins (phosphorylated VASP serine 157/239 and phosphorylated Zyxin serine 142). VASP silencing in K562 cells resulted in a 40% decrease of CD61 expression at the end of the megakaryocyte differentiation (P<0.05), whereas Zyxin silencing resulted in a 15% decrease of CD41 expression (P<0.01). VASP expression was reduced during Imatinib treatment of K562 cells, as was also its interaction with BCR-ABL protein. In addition, VASP silencing resulted in a decrease of FAK phosphorylation, an effector of the BCR-ABL pathway involved in cellular adhesion of K562 cells. Conclusions: VASP and Zyxin proteins have a role in hematopoiesis, including megakaryocyte differentiation. Alterations in VASP and Zyxin expression affect differentiation and apoptosis of hematopoietic cells. VASP may participate in the BCR-ABL signaling pathway of leukemic cells, affecting leukemic cell adhesion through FAK activity. The elucidation of VASP and Zyxin functions will help elucidate the mechanisms of hematopoietic disorders, such as CML and others. Disclosures: No relevant conflicts of interest to declare.

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The Role of Hypoxia on ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Blood ◽

10.1182/blood-2019-129768 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5369-5369

Author(s):

Seiichi Okabe ◽

Yuko Tanaka ◽

Mitsuru Moriyama ◽

Akihiko Gotoh

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

K562 Cells ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Transfected Cells ◽

Resistant Cells

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Moreover, ABL TKIs cannot cure the Ph-positive leukemia patients because of leukemia stem cells in the bone marrow niche. Therefore, new approach against leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Hypoxia is an important component of the bone marrow microenvironment. Because oxygen tension plays a key role in driving normal hematopoiesis, leukemia stem cells may be maintained in hypoxic areas of the bone marrow. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R and Ba/F3 T315I). We investigated gene expression profiles in cultured ABL TKI resistant cells and parental cell line, K562 in normoxia and hypoxia condition by DNA microarray. Results: We first investigated gene expression profiles in cultured K562 cells in hypoxia condition. We found gene expression of insulin-like growth factor 1 (IGF1) was increased K562 cells in hypoxia condition by DNA microarray. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. We could not detect the BCR-ABL point mutation in ABL TKI resistant cells. We found gene expression of insulin-like growth factor 1 (IGF1) receptor (IGF1R) was increased ABL TKI resistant K562 cells. IGF1R gene amplification was confirmed by RT-PCR analysis. IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). One of IGFBP, IGFBP5 is related to imatinib sensitivity and resistant in chronic myeloid leukemia (CML) patients (GSE12211). In hypoxia condition, several IGFBPs were also increased in ABL TKI resistant cells. IGF cause intracellular signaling that ultimately results in cellular growth and proliferation. Thus, we initially examined whether addition of IGF1R inhibition could enhance ABL TKIs sensitivity. One of IGF1R inhibitor, linsitinib was inhibited ABL TKI resistant cells and parental cell line, K562 in hypoxia condition. ABL TKI resistant cell lines were more sensitive against linsitinib. Combined treatment of ABL TKI resistant cells and K562 cells with ABL TKIs and linsitinib caused more cytotoxicity than each drug alone in hypoxia condition. Caspase 3/7 activity and cellular cytotoxicity was also increased after ABL TKIs and linsitnib treatment. In the colony formation method, the number of cell colonies were also reduced in hypoxia condition. Intracellular ATP levels have been implicated in vitro as a determinant of cell death by apoptosis. The concentrations of intracellular ATP were reduced after ABL TKIs and linsitinib. We next blocked IGF1R function by small interfering RNA (siRNA). SiRNA transfected cells were reduced cellular proliferation. We also found drug sensitivity of the cells to the imatinib was increased compared to mock-transfected cells. Apoptotic cells and caspase 3/7 activity were increased after imatinib treatment in siRNA transfected cells. Conclusion: The IGF1 pathway is involved in Ph-positive leukemia cells in hypoxia condition and ABL TKI resistant in CML cells. We also provide the promising clinical relevance as a candidate drug for treatment of residual leukemia cells in bone marrow niche which is in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

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Evaluation of Ph+/CD34+ Residual Cells in Chronic Myeloid Leukemia Patients after Long Lasting Treatment with Imatinib Mesylate.

Blood ◽

10.1182/blood.v106.11.1999.1999 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1999-1999

Author(s):

Monica Bocchia ◽

Elisabetta Abruzzese ◽

Micaela Ippoliti ◽

Simona Calabrese ◽

Alessandro Gozzetti ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Imatinib Mesylate ◽

Median Time ◽

False Positive ◽

Myeloid Leukemia ◽

Prolonged Treatment ◽

Published Data ◽

Imatinib Treatment ◽

Cd34 Cells

Abstract Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES<1% false positive; bcr-abl Dual Fusion 8–10% false positive) The clinical significance of these data as well as the role of this cell target to monitor minimal residual disease in CML needs to be evaluated on a larger serie of patients.

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Preliminary Study On Influence of Imatinib-Induced Apoptosis in Chronic Myeloid Leukemia Cells Co-Culture with Fibronectin Adhesion.

Blood ◽

10.1182/blood.v114.22.4264.4264 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4264-4264

Author(s):

Liu Xiaoli ◽

Na Xu ◽

Qingfeng Du ◽

Zhi Liu ◽

Rong Li ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Growth Inhibition ◽

Myeloid Leukemia ◽

K562 Cells ◽

Bone Marrow Microenvironment ◽

Dependent Manner ◽

Imatinib Resistance ◽

Significant Difference

Abstract Abstract 4264 The bone marrow microenvironment supports growth, differentiation and proliferation of normal hematopoietic cells and can also contribute to malignant growth. Recently,it is considered that except for the point mutant of BCR-ABL kinase contribute to imatinib-resistant therapy for patients with chronic myeloid leukemia(CML), environment-mediated drug resistance (EM-DR) is a potential factor in imatinib resistance. Our previous studies found that Integrin, focal adhesion kinase(FAK), RhoA(a small GTPase) are important adhesion molecules,and related to imatinib resistance. But how and what they crosstalk with each other is still open to debate. In order to simulated bone marrow microenvironment, we used the major components of bone marrow microenvironment- Fibronectin (Fn) co-cultured with human leukemia K562 cells.and then K562 cells were inoculated with Fn, collagen-coated plate(Co) and suspended cultures as control(mask) group,and then treated with 0.4μM,0.8μM,1.6μM,3.2μM,6.4μM imatinib for 24h,48h and 72h, detected cell apoptosis and proliferation by MTT and AnnexinV-PI assay, examined p-FAK and Rho-GTP by Wersten Blotting and Pull down-Wersten Blotting. The data showed that compared to the Co and mask groups, the cells growth inhibition and apoptosis in Fn co-culture group was significantly reduced. The protein expression of p-FAK and Rho-GTP was higher in the Fn group,and in time-dependent manner. When K562 cells in Fn group were transfected with 150nM siRNA-RhoA for 48h, there was no significant difference compared with the Co and mask groups. Furthermore, the above groups treated with anti-integrin monoclonal antibody (anti-CD29 mAb), we found that p-FAK was significant lower compared with without anti-CD29 mAb in the Fn group; but there was no significant difference of Rho-GTP compard with without anti-CD29 mAb in Fn group. These results indicate that Fn adhesion co-culture could reduce imatinib-induced cell growth inhibition and apoptosis, and this mechanism may be correlated to Rho-GTP activity,and anti-integrin monoclonal antibody could not completely block the integrin binding to Fn on K562 cells, or there was other pathway activated RhoA. The mechanism of EM-DR is complex,and which is well worth us to speculate and study. Disclosures: No relevant conflicts of interest to declare.

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Role of Folate Receptor 3 in Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-111611 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2998-2998

Author(s):

Na Shen ◽

Xiaojian Zhu ◽

Shu Zhou ◽

Yong You ◽

Zhaodong Zhong ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Chronic Myeloid Leukemia ◽

Tumor Cell ◽

Transcriptome Analysis ◽

Myeloid Leukemia ◽

Folate Receptor ◽

K562 Cells ◽

Tumor Cell Differentiation

Abstract Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by Philadelphia chromosome and its molecular counterpart, the BCR-ABL1 fusion gene. Discontinuation of tyrosine kinase inhibitors (TKIs) therapy after achieving a persistent deep molecular response (DMR) is an urgently-needed treatment goal for patients and it was written in NCCN guideline Version 2.2017 for CML. Indeed, various studies have confirmed the feasibility of stopping TKIs therapy in many regions. However, increasing evidence demonstrated that TKIs are unable to eliminate quiescent leukemic stem cells (LSCs), which lead to treatment resistance or relapse after discontinuation of TKIs treatment. Multiple works have confirmed that approximately 50-60% of DMR patients stopping TKIs will lose their response and require retreatment. The patients who will lose the treatment-free remission (TFR) remains unknown currently. We selected bone marrow of 14 patients (matched 7 relapse and 7 non-relapse patients) who discontinued TKIs therapy in our center for transcriptome analysis to discover the differences between relapse and non-relapse patients. Bioinformatics indicated that folate receptor 3 (FOLR3) was highly expressed in non-relapse patients and only in the non-relapsers (Figure 1A). Further work found these samples all carried a common SNP mutation. Then we respectively transferred newly diagnosed chronic phase (CP) CML patients derived bone marrow CD34+ cells and CML K562 cells with lentiviral vectors containing FOLR3 SNP, FOLR3 SNP shRNA, and wild type FOLR3. The result indicated that FOLR3 SNP significantly promoted the clonogenicity of CD34+/K562 cells, while loss of FOLR3 SNP hindered cell differentiation (Figure 1B and D are data of CD34, Figure 1C and E are data of K562). K562 subcutaneous tumor formation in balb/c node mice confirmed that tumors weight and volume of FOLR3 SNP shRNA group were higher than control. While, small animal PET scanning showed that maximum standardized uptake value (SUVmax) of 18F-FDG of FOLR3 SNP+ group was higher than the rest. Experiments in vivo and vitro synergistically proved FOLR3 SNP promoted tumor cell differentiation, delayed tumor growth. To better understand how FOLR3 SNP promote tumor cell differentiation and delay tumor growth, we performed second transcriptome analysis. Consistently, both in FOLR3 SNP+ CD34+ and K562 cells, enrichment analysis revealed that differentially expressed genes were enriched in mitochondria associated Gene Ontology (GO) biological process (Figure F), in which mitochondria complex V matched genes were most significant, such as ATP5 family. In vitro, we demonstrated that ATP syntheses, maximal respiration and spare respiratory capacity of FOLR3 SNP+ CD34+/K562 cells were significantly higher than control and shFOLR3 counterpart through seahorse XF cell mito stress test (Figure G and H refer to CD34 and K562, separately). Electron microscope also exhibited an increase of mitochondrial in FOLR3 SNP+ cells. Mitochondria play an essential role in energy generation, cell signalling, differentiation, death and senescence in eukaryotic cells. We detected a series of genes related to aging, cell cycle and mitochondria unfolded protein response. The results showed that cell cycle kinase such as CDK4 decreased in FOLR3 SNP+ CD34+/K562 cells. On the other hand, senescence associated genes seemed increased. In conclusion, we highlighted the connection of FOLR3 and post-cessation relapse. FOLR3 SNP could be an indicator of TFR. Its internal mechanism might be the mitochondrion activation induced aging of residual leukemia cells. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Overexpression of the EphB4 Receptor Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Through Regulating Mlcp and VAV1

Blood ◽

10.1182/blood.v120.21.4421.4421 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4421-4421

Author(s):

Liu Xiaoli ◽

Jinfang Zhang ◽

Qingfeng Du ◽

Na Xu ◽

Lulu Xu ◽

...

Keyword(s):

Cell Adhesion ◽

Chronic Myeloid Leukemia ◽

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Myeloid Leukemia ◽

K562 Cells ◽

Differential Expression Genes

Abstract Abstract 4421 Objective: To study the role of EphB4 in imatinib (IM) resistant chronic myeloid leukemia (CML) and investigate the mechanism. Methods: We derived IM-resistant cells, K562-R cells, from wild K562 cells under gradually increasing IM concentrations. We analysed expression level of EphB4 in CML patients, wild K562 and K562-R cell lines by real-time reverse transcription PCR and Western blot analysis. Then we established stable under-expressing EphB4 cell (K562-R-EphB4-sh) lines. We analysed the sensitive for IM of K562, K562-R, K562-R-EphB4-sh cell lines by CCK8 assay. Microarray analysis was used to screen differential expression genes between K562-R and K562-R-EphB4-sh cell lines. Results: The mRNA and protein of EphB4 were significantly increased in IM resistant CML patients compared to IM sensitive CML patients (p<0.05). The Similar results were observed in K562-R and K562 cells (p<0.01). To analyze the role of EphB4 in IM resistance, EphB4 was knocked down with shRNA expressed by pLL3.7 lentivirus vector. We established stable under-expressing EphB4 cell line K562-R-EphB4-sh. RT-PCR and western blot analysis showed that mRNA and protein expression of EphB4 in K562-R-EphB4-sh cells were reduced (p<0.05). CCK8 assay found K562 cells (IC50 0.1207±0.0234μM), K562-R-EphB4-sh cells (IC50 0.7228±0.04752μM) were sensitive to IM but K562-R (IC50 2.8101±0.04674μM) still showed IM resistance (p<0.05). Those suggested K562-R-EphB4-sh cells resensitize to IM when the expression of EphB4 was down regulated. However, these cells were still less sensitive than K562 cells. Microarray analysis between K562-R and K562-R-EphB4-sh cell lines found 641 differential expression genes, most of them were related to cell adhesion and cell cytoskeleton. We confirmed MLCP and VAV1 were down regulated in K562-R-EphB4-sh cells compared to K562-R cell lines by western blot analysis. Conclusion: Our study suggest EphB4 receptor contributes to IM-resistant in CML through regulating cell adhesion molecular MLCP and VAV1, which may provide new biomarkers and contribute to] developping new drugs for the disease. Disclosures: No relevant conflicts of interest to declare.

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FMNL1 Is Downregulated In Myelodysplastic Syndromes and Is Involved In Megakaryocytic Differentiation

Blood ◽

10.1182/blood.v122.21.1519.1519 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1519-1519

Author(s):

Matheus Rodrigues Lopes ◽

João Agostinho Machado-Neto ◽

Fabiola Traina ◽

João Kleber Novais Pereira ◽

Irene Lorand-Metze ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Bone Marrow Cells ◽

K562 Cells ◽

Fold Increase ◽

Granulocytic Differentiation ◽

Megakaryocytic Differentiation ◽

Total Bone Marrow ◽

Related Proteins ◽

Dependent Processes

Abstract Background Myelodysplastic syndromes (MDS) are disorders characterized by morphological dysplasia, impaired differentiation and defective cellular functions, resulting in peripheral cytopenias. FMNL1 belongs to a family of formin-related proteins, indispensable for many fundamental actin-dependent processes. Recently, FMNL1 has been described to be upregulated and play a role in the actin cytoskeleton dynamics during monocyte differentiation to macrophages. Aims The aim of this work was to characterize FMNL1 expression in total bone marrow cells of patients with MDS comparing to normal donors. We also analyzed FMNL1 expression in erythrocytic, granulocytic and megakaryocytic differentiation, using cell line models. Finally, we evaluated the impact of inhibition of FMNL1 during megakaryocytic differentiation. Methods A total of 49 patients with a diagnosis of MDS, receiving no treatment, and 18 samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. Samples were submitted to RNA extraction after removal of erythrocytes by hemolysis. FMNL1 expression levels from cell lines or total bone marrow cells were determined by quantitative PCR (q-PCR) or Western blot. KU812 was treated 50 μM hemin and 100 μM hydroxyurea for erythrocytic differentiation. K562 was stimulated with 20nM of PMA for megakaryocytic differentiation. NB4 was treated with 10-6 M of ATRA for granulocytic differentiation. Megakaryocytic differentiation was followed by the increase in megakaryocytic marker (CD61) determined by flow cytometry and cells were also stained with May–Grunwald–Giemsa. K562 cells were transduced with lentivirus-mediated shRNA targeting LacZ or FMNL1. Apoptosis was assessed by Annexin-V/PI staining and cell cycle was evaluated by flow cytometry, both at 24 and 48 hours after induction with PHA. The statistical methods used were the age-adjusted multivariate linear regression analysis, Mann Whitney test or t test. Results FMNL1 expression in bone marrow samples was significantly lower in MDS when compared with normal donor cells (P=0.01), especially in the high risk group (P<0.02). Using cell line models for hematopoietic differentiation, there was a fifteen-fold increase and a five-fold increase in FMNL1 expression for megakaryocytic (P=0.002) and granulocytic differentiation (P=0.05) respectively. Western blot analysis corroborated these findings. There was no difference for erythrocytic differentiation. After PMA treatment, the level of the megakaryocytic markers CD61 was significantly lower in K562 shFMNL1 when compared with shLacZ (P=0.01). The level of CD41a (P=0.5) and CD42b (P=0.1) showed a trend toward a decrease in K562 shFMNL1 when compared with shLacZ, however not statistically significantly. Corroborating these data, K562 shFMNL1 showed abnormal megakaryocytic morphological features (larger cells with polylobulated or polysegmented nuclei and vacuolization) compared with cells shLacZ. There were no statistical differences in the apoptosis levels and cell cycle analysis between K562 shFMNL1 and shLacz cells. Conclusions The family of formin-related proteins has mainly been related to actin-dependent processes although little is known regarding their possible involvement in haematopoiesis. The lower FMNL1 expression in MDS BM could reflect the role of this protein in cell differentiation. We were therefore prompted to study this issue in depth using megakaryocytic differentiation as a system. In this study, we show increased expression of FMNL1 in PMA-induced megakaryocytic differentiation of K562 cells. Furthermore, knockdown of FMNL1 deregulates differentiation, suggesting that FMNL1 is required in order to maintain the effective megakaryocytopoiesis in MDS. Although FMNL1 silencing effectively down-regulated CD61 expression, CD41a and CD42b were reduced to a lesser extent. The effect of FMNL1 cannot be explained by modifications of the cell cycle or apoptosis during differentiation; and is probably due to the effect in changes in the dynamic remodeling of the cytoskeleton. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of cystathionine β-synthase promotes apoptosis and reduces cell proliferation in chronic myeloid leukemia

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00410-5 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Dan Wang ◽

Huan Yang ◽

Yun Zhang ◽

Rong Hu ◽

Dongjie Hu ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

K562 Cells ◽

Bone Marrow Mononuclear Cells ◽

Cycle Arrest ◽

Primary Bone ◽

And Migration

AbstractIncreased endogenous hydrogen sulfide (H2S) level by cystathionine β-synthase (CBS) has been shown to closely relate tumorigenesis. H2S promotes angiogenesis, stimulates bioenergy metabolism and inhibits selective phosphatases. However, the role of CBS and H2S in chronic myeloid leukemia (CML) remains elusive. In this study, we found that CBS and H2S levels were increased in the bone marrow mononuclear cells of pediatric CML patients, as well as in the CML-derived K562 cells and CBS expression levels were correlated with different disease phases. Inhibition of CBS reduced the proliferation of the CML primary bone marrow mononuclear cells and induced growth inhibition, apoptosis, cell cycle arrest, and migration suppression in K562 cells and tumor xenografts. The knockdown of CBS expression by shRNA and inhibiting CBS activity by AOAA decreased the endogenous H2S levels, promoted mitochondrial-related apoptosis and inhibited the NF-κB-mediated gene expression. Our study suggests that inhibition of CBS induces cell apoptosis, as well as limits cell proliferation and migration, a potential target for the treatment of chronic myeloid leukemia.

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The Importance of Bone Marrow Lymphocyte Subtypes As Predicting Factors for Molecular Recurrence in Patients with Chronic Myeloid Leukemia after Discontinuation of Imatinib

Blood ◽

10.1182/blood-2021-149242 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2564-2564

Author(s):

Arthur Gomes Oliveira Braga ◽

Katia B Pagnano ◽

Marina Dal'Bó Pelegrini Campioni ◽

Ana Beatriz P Lopez ◽

Konradin Metze ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Board Of Directors ◽

Median Time ◽

Myeloid Leukemia ◽

Lymphocyte Subsets ◽

Chronic Phase ◽

Imatinib Treatment ◽

Advisory Committees ◽

Treatment Free Remission

Abstract Introduction: in recent years the feasibility of the discontinuation of tyrosine kinase treatment in chronic myeloid leukemia (CML) has been proven, and several clinical factors influencing the duration of treatment-free remission (TFR) after discontinuation have been studied. Aim: we analyzed the influence of bone marrow (BM) lymphocyte subsets on the molecular recurrence after discontinuation of Imatinib (IM) in CML. Methods: in a recent discontinuation study performed at our Institution (EDI-PIO trial) we assessed BM lymphocyte subsets before and after introduction of pioglitazone which was given 3 months before discontinuation of IM. Criterias for discontinuation were: patient in chronic phase at diagnosis, a minimum of 3 years on TKI and sustained molecular remission MR4.5 for at least 2 years. Lymphocyte populations studied: B, TCD8, TCD4, T CD4+CD8+ and T CD4- CD8- besides T naïve and memory, TCRαβ, TCRγδ, NK-t and NK cells. The influence of Sokal score at diagnosis, the duration of imatinib treatment together with the BM lymphoid subsets on the time of treatment-free remission (TFR) were examined by uni- and multivariate Cox regressions. Results: we studied 30 out of 32 patients diagnosed between 1998 and 2013 that reached criteria for discontinuation that were included in EDI-PIO trial: 13 male and 17 female. Median age at diagnosis: 41 years (22-65). Median time of IM treatment: 116.3 months (38.2-209.3); 11 patients (36%) had a molecular recurrence in a median of 5,17 months (2.4-29.4). For patients remaining in TFR the median time of follow-up was 46 months (26.3-55.9). Median overall time of IM treatment (IM-Tr) was 65 months for patients recurring and 124 months for those remaining in TFR. In the univariate Cox regression a significant value was found for IM-Tr, higher percentages of T CD4+CD8+ and lower ones of TCRγδ lymphocytes at discontinuation. In the multivariate model only the T CD4+CD8+ remained. Conclusion: discontinuation of IM is feasible in patients remaining in continuous MR4.5 for more than 2 years. A longer time of IM treatment and higher values of BM T CD4+CD8+ predict a lower risk of relapse. Key words: chronic myeloid leukemia, imatinib, discontinuation, lymphocytes, pioglitazone Disclosures Pagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture.

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