scholarly journals The GPR68/BCL2 Axis Remodels Metabolism in AML By Relocating Calcium

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2661-2661
Author(s):  
Xiaofei He ◽  
Caleb Hawkins ◽  
Lauren Lawley ◽  
Mark Wunderlich ◽  
Benjamin Mizukawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Survival ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Tca Cycle ◽  
Cooperative Effect ◽  
O2 Consumption ◽  
Hematopoietic Stem ◽  
Atp Production

Hematopoietic stem cells (HSC) mainly engage glycolysis while leukemia stem cells (LSC), such as in acute myeloid leukemia (AML), heavily rely on mitochondrial (Mt) respiration (i.e. oxidative phosphorylation, OxPhos) to fuel energy. Growing evidence suggests this metabolic reprogramming confers therapeutic vulnerabilities in AML. BCL2 is overexpressed in LSC from AML patients, while BCL2 inhibitors, such as venetoclax (VEN), have been shown to suppress OxPhos in LSC and to eradicate LSC. In clinical practice, half of the patients fail to respond to VEN. VEN is a costly medication and patients who are resistant to VEN forgo alternative treatment at that time. Prediction of the response to VEN and strategies to circumvent resistance are urgently needed. BCL2 is shown to increase Mt Ca2+ levels ([Ca2+]m), which enhance OxPhos through activation of the Ca2+-sensitive dehydrogenases within the tricarboxylic acid (TCA) cycle. G protein-coupled receptor 68 (GPR68) is a proton sensor, activating phospholipase C that leads to releasing of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and elevation of cytosolic Ca2+ levels ([Ca2+]c). This prompted us to examine the cooperative effect of GPR68 and BCL2 on the Ca2+/OxPhos pathway in AML, and particularly in LSC. Expression of leukemic oncogenes (i.e. MLL-AF9 and HRASG12D) in mouse hematopoietic stem and progenitor cells, such as Lineage-Sca-1+cKit+ (LSK) cells or granulocyte-monocyte progenitor cells, promote leukemogenesis as evidenced by serial colony formation in vitro and leukemia development in vivo. We found significantly reduced colonies in oncogene-expressing LSK cells from Gpr68 knockout mice compared to wild type mice. Deletion of Gpr68 reduced [Ca2+]c in oncogene-expressing LSK cells. We next examined the function of GPR68 in human AML cell lines. Knockdown of GPR68 with shRNA reduced cell growth and colony formation, and induced apoptosis in AML cells. Knockdown of GPR68 also reduced [Ca2+]c and Mt membrane potential (Δψm) in AML cells, indicating reduced Mt OxPhos. These results suggest that GPR68 regulates the Ca2+/OxPhos pathway in AML cells. We next examined the cooperative effect of GPR68 and BCL2 by jointly inhibiting their activities with pharmacological agents (i.e. GPR68 antibody and VEN, respectively) in AML cell lines and AML patient-derived xenograft models. Of note, the expression of GPR68 was positively correlated with the sensitivity to VEN in AML cells. For AML cells that were resistant to VEN, GPR68 antibody but not an unrelated antibody increased the sensitivity to VEN by enhancing apoptosis, indicating that GPR68 and BCL2 co-regulate AML cell survival. We next examined the mechanism of this synthetic lethality by measuring cellular respiration. Single treatment with VEN reduced Δψm, ATP production and O2 consumption in AML cells. Cotreatment with VEN and GPR68 antibody further reduced Δψm, ATP production and O2 consumption in AML cells, indicating that GPR68 and BCL2 co-regulate Mt OxPhos. Given that GPR68 releases Ca2+ from ER to cytosol, while BCL2 maintains [Ca2+]m by inhibiting its extrusion from Mt, we hypothesize that the GPR68/BCL2 axis relocates Ca2+ from ER to Mt. As expected, treatment with GPR68 antibody reduced [Ca2+]c. Cotreatment with VEN and GPR68 antibody increased [Ca2+]c, indicating enhanced extrusion of Ca2+ from Mt to cytosol by VEN. Consistently, cotreatment with VEN and GPR68 antibody reduced the activity of isocitrate dehydrogenase, the rate limiting enzyme in the TCA cycle, in AML cells. These results indicate that GPR68 and BCL2 co-regulate the Ca2+/OxPhos pathway in AML cells and that co-inhibition of GPR68 and BCL2 may enhance lethality and overcome VEN resistance. In summary, our study suggests that the GPR68/BCL2 axis co-regulates AML cell survival by relocating Ca2+ from ER to Mt thus enhancing Mt OxPhos, and that disruption of the GPR68/BCL2 axis provides a novel therapeutic strategy to overcome resistance to VEN. Disclosures No relevant conflicts of interest to declare.

CD52 Expression In Leukemic Stem/Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2743-2743 ◽  
Author(s):  
Vivian G. Oehler ◽  
Roland B. Walter ◽  
Carrie Cummings ◽  
Olga Sala-Torra ◽  
Derek L. Stirewalt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Cell Populations ◽  
Cell Surface Glycoprotein ◽  
Hematopoietic Stem

Abstract Abstract 2743 CD52 is a cell surface glycoprotein of unknown function that is expressed in B and T lymphocytes, macrophages, and monocytes, but is not expressed in normal hematopoietic stem/progenitor cells. CD52 is also expressed in chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (ALL), and some cases of T-ALL. Alemtuzumab, a recombinant humanized monoclonal antibody, targets CD52 and is used to treat CLL. In contrast to normal hematopoietic stem/progenitor cells, CD52 expression has been described in acute myeloid leukemia (AML) and in blast crisis (BC) chronic myeloid leukemia (CML). Based on these observations we were curious whether CD52 expression distinguished normal from malignant or more mature from immature stem/progenitors cells, and whether these cells were sensitive to alemtuzumab. CD52 expression was examined in three blast cell populations (CD34+/CD38-, CD34+/CD38+, and CD34-) in patients with myeloid (44) and lymphoid (18) neoplasms, and normal patients (6). In normal hematopoietic cells, stems cells are enriched in the first population; more mature cells are characterized by increasing CD38 expression and loss of CD34 expression. In AML and CML leukemia stem cells may arise within either CD34+ population and possibly in the CD34- population. Relative to normal lymphocytes average CD52 expression could be characterized as low to moderate. Using an expression cutoff of > 20%, in contrast to normal patients, CD52 was detected in at least one of three blast populations in almost all patients. Using a more stringent cutoff of > 50%, CD52 was expressed in CD34+/CD38- cells in 7/11 B-ALL and 6/7 T-ALL cases and was concordantly expressed in the other two populations. Using the same criteria in myeloid malignancies (Table 1), expression occurred more frequently in AML, AML arising from myelodysplastic syndrome (MDS), and BC CML. In AML and AML arising from MDS, CD52 was expressed in the 34+/38- population in 7/15 cases (47%) and 4/7 cases (57%), respectively; it was expressed in both BC CML patients. In AML and BC CML patients, CD52 was expressed at similar levels in the CD34+/CD38+ fraction. No clear association between CD52 expression and cytogenetic abnormalities was found. We then examined whether CD52 expression differentiated normal from malignant blasts (CD34+/CD38- and CD34+/CD38+) in two CML myeloid BC patients. FISH and quantitative PCR demonstrated that BCR-ABL was expressed in all 4 populations, which were also morphologically distinct. Colony forming unit (CFU) assays demonstrated a significantly decreased ability to form CFU (on average 5–20 fold decrease) in CD52+/CD34+/CD38- CML cells suggesting CD52 cells may be more mature. Lastly and not previously described, we found that several BC CML cell lines express CD52, and complement-mediated cell cytotoxicity was similar in the highest expressing cell lines to that seen in EHEB (B-CLL) cells known to be targeted by alemtuzumab. Thus, alemtuzumab may have clinical efficacy in BC CML. In conclusion, CD52 is expressed on blast populations enriched for leukemic stem cells. Whether the absence or presence of CD52 more precisely segregates a leukemia stem cell containing population currently remains unknown and requires functional testing in a murine model. Our preliminary experiments in CML suggest CD52 may not differentiate between normal and malignant stem/progenitor cells. However, CD52 expression may distinguish normal and malignant stem cell populations in cases where CD52 and CD38 are more highly expressed. The observation that CD52 expression is increased in acute vs. chronic leukemias raises the intriguing possibility that CD52, if not directly involved, may be a marker for genes or pathways contributing to the block in differentiation seen with progression to acute leukemia. Furthermore, given that CD52 expression is heterogeneous in chronic disorders, it is possible that CD52 expression within these populations may correlate with poor prognosis or impending leukemic conversion. Table 1. The proportion of patients (44) expressing CD52 at levels > 50% in 3 blast populations. Three populations were present in most, but not all patients. Gray shading indicates chronic myeloid diseases. MPN is myeloproliferative neoplasm; NOS is not otherwise specified; ET is essential thrombocythemia; CMML is chronic myelomonocytic leukemia; and an arrow represents progressed to. Disclosure: Oehler: Pfizer: Research Funding. Radich:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3764-3764
Author(s):  
Patali S Cheruku ◽  
Marina Bousquet ◽  
Guoqing Zhang ◽  
Guangtao Ge ◽  
Wei Ying ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Gene Profiling ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Signature Genes

Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3750-3750
Author(s):  
Angeliki Thanasopoulou ◽  
Katharina Dumrese ◽  
Sarah Picaud ◽  
Oleg Fedorov ◽  
Stefan Knapp ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines

Abstract The CBP/p300 histone acetyltransferases are key transcriptional regulators of hematopoiesis that have been found to be involved in AML-associated recurrent chromosomal translocations and shown to function as co-activators of leukemogenic fusion oncogenes, suggesting that specific targeting of CBP/p300 may be beneficial for therapy. We characterized the anti-leukemic potential of I-CBP112, a novel chemical inhibitory probe targeting the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. I-CBP112 represents a new, potent and selective class of BRD inhibitors (oxazepines) binding to recombinant CBP/p300 BRDs with a KD of 151nM and 157nM respectively. Initial characterization by FRAP and BRET assays revealed that I-CBP112 displaced the isolated BRD construct from chromatin but not the full length CBP. I-CBP112 also impaired the interaction of CBP/p300 with p53, resulting in reduced p53-K382 acetylation, reduced p21 expression, and high sensitivity to Doxorubicin-induced DNA damage. We started to explore the effects of the compound on leukemic cells by exposing a series of murine cell lines immortalized by the MLL-CBP fusion and other potent leukemia-associated oncogenes including the MLL-AF9, MLL-ENL, or the NUP98-HOXA9 fusion to increasing doses of I-CBP112. Interestingly, no significant cytotoxicity was observed up to concentrations of 5μM. However, in all cell lines we observed a significant reduced number of colonies formed in methylcellulose, associated with morphological differentiation as observed in Giemsa stained cytospots. Similar to the murine leukemic cell lines we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Reduced colony formation upon I-CBP112 treatment was also observed of human primary AML blasts but not of CD34+ hematopoietic stem cells from two healthy donors. I-CBP112 effects were studied in more detail in three human leukemia cell lines: SEM (MLL-AF4+), MOLM13 (MLL-AF9+) and Kasumi-1 (AML1-ETO+). Long-term exposure of these cells to I-CBP112 in liquid medium, resulted in a dose-dependent G1 cell cycle arrest, with Kasumi-1 being the most sensitive to the inhibitor, demonstrating further morphological signs of differentiation and apoptotic cell death. Importantly, combination of I-CBP112 with the BET-BRD inhibitor JQ1 or Doxorubicin revealed a clear synergistic effect on cell survival of the AML cell lines except for the combination of I-CBP112 with Doxorubicin on MOLM13. Surprisingly only modest effects of I-CBP112 exposure on the transcriptional programs of SEM, MOLM13 and Kasumi-1 cells were found by microarray expression profiling. Genes found affected were mainly immune response regulators or NFkappaB targets suggesting that attenuation of NFkappaB downstream signals might impair the leukemia initiation capacity reflected by reduced colony formation. Extreme limited dilution assays (ELDA) in methylcellulose, as well as bone marrow transplantations in limiting dilutions using MLL-AF9-transformed murine leukemic blasts revealed that I-CBP112 significantly impaired self-renewal of the leukemic stem cell compartment in vitro and reduced the leukemia-initiating potential in vivo. Taken together, these data demonstrate that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML and other human cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Growth-Supporting Activities of Fibronectin on Hematopoietic Stem/Progenitor Cells In Vitro and In Vivo: Structural Requirement for Fibronectin Activities of CS1 and Cell-Binding Domains

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3263-3272 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Hideki Mitsui ◽  
Keisuke Aoyama ◽  
Jun Ishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Cell Binding ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Colony Forming Units

Abstract Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34+/VLA4bright/VLA5dullhematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor–deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34+ cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34+ cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.

scholarly journals CD98: a type II transmembrane glycoprotein expressed from the beginning of primitive and definitive hematopoiesis may play a critical role in the development of hematopoietic cells

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3676-3687 ◽  
Author(s):  
AP Warren ◽  
K Patel ◽  
DJ McConkey ◽  
R Palacios
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Survival ◽  
Progenitor Cells ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Cloning And Expression ◽  
Type Ii ◽  
Hematopoietic Stem

In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.

scholarly journals Growth-Supporting Activities of Fibronectin on Hematopoietic Stem/Progenitor Cells In Vitro and In Vivo: Structural Requirement for Fibronectin Activities of CS1 and Cell-Binding Domains

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3263-3272 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Hideki Mitsui ◽  
Keisuke Aoyama ◽  
Jun Ishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Cell Binding ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Colony Forming Units

Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34+/VLA4bright/VLA5dullhematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor–deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34+ cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34+ cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.

scholarly journals Overcoming the effect of ASH1L haploinsufficiency on stem cells amenability to genome editing and differentiation into the neuronal lineage – a technical report

2021 ◽  
Author(s):  
Seon Hye Cheon ◽  
Foster D Ritchie ◽  
Janay M Vacharasin ◽  
Nicholas Marinelli ◽  
Collin Cheatle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Survival ◽  
Genome Editing ◽  
Neuronal Differentiation ◽  
Cell Lines ◽  
Autism Spectrum ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Technical Report

Genome editing and neuronal differentiation protocols have proliferated in the last decade. Mutations in genes that control pluripotency could lead to a potential obstacle with regards to the survival and differentiation potential of the genome-edited cell lines. Here we describe a protocol for the generation, and differentiation, of cell lines containing CRISPR/Cas9 induced mutations in the histone methyltransferase ASH1L. This chromatin modifier was previously implicated in hematopoietic stem cell pluripotency and is a major genetic risk factor for autism spectrum disorders (ASD). We find that haploinsufficiency of ASH1L leads to decreased NANOG gene expression leading to reduce cell survival and increased spontaneous differentiation. We report a method that provides improved single-cell survival with higher colony formation efficiency in ASH1L mutant stem cells. Additionally, we describe a modified dual-SMAD inhibition neuronal induction methodology that permits the successful generation of human neurons with mutations in ASH1L, in a smaller scale than previously reported methods. With our modified CRISPR-genome editing and neuronal differentiation protocols, it is possible to generate genome-edited stem cells containing mutations in genes that impact pluripotency and could affect subsequent cell lineage-specific differentiation. Our detailed technical report presents cost-effective strategies that will benefit researchers focusing on both translational and basic science using stem cell experimental systems.

Targeting the CML Stem/Progenitor Cell with JAK2 (BMS-911543)/ABL Inhibitor Combination Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2743-2743
Author(s):  
Hanyang Lin ◽  
Matthew Lorenzi ◽  
Adrian Woolfson ◽  
Xiaoyan Jiang
Keyword(s):  
Combination Therapy ◽  
Western Blot ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Combination Treatment ◽  
Blot Analysis ◽  
Colony Formation ◽  
K562 Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2743 The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. The first specific BCR-ABL inhibitor, Imatinib Mesylate (IM), has had a major impact on the treatment of chronic phase CML. However, early relapse and molecular evidence of residual disease remain a significant issue in some IM-treated patients. We and others have recently demonstrated that CML stem/progenitor cells are insensitive to IM, genetically unstable, and characterized by elevated BCR-ABL expression and TK activity. The prevention of the development of resistant subclones through the specific targeting of CML stem/progenitor cells consequently represents a key unmet medical need in CML therapy. We recently identified the presence of a physical complex between oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and JAK2. This molecular complex contributes to the transforming activity of BCR-ABL and plays a key role in the IM responsivity/resistance of primary CML stem/progenitor cells. We therefore hypothesized that a combination treatment strategy designed to destabilize the complex might provide a rational approach to the elimination of CML stem/progenitor cells. Several JAK2 inhibitors are currently in various stages of clinical development, but myelosuppressive effects of these inhibitors on normal hematopoietic stem/progenitor cells remains a concern. We have examined a new and selective JAK2 inhibitor (BMS-911543) in combination with a number of different tyrosine kinase inhibitors (TKIs), including IM, dasatinib (DA), and nilotinib (NL) in CML cells. First, Western blot analysis showed that combination treatment with IM and BMS-911543 was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells (K562R) than in cells treated with BMS-911543 or IM alone. BMS-911543 treatment alone was not though effective in reducing pSTAT5 levels. Consistent with the Western blot analysis, in colony-forming cell (CFC) assays CML cell lines derived from myeloid or B-lymphoid blast crisis patient samples that expressed high levels of BCR-ABL (K562, K562R, BV173, and UT7 transduced with BCR-ABL) demonstrated reduced colony numbers, especially in medium-sized colonies (50–500 cells per colony) following combination treatment (BMS-911543 and IM) as compared with cells treated with BMS-911543 or IM alone (∼2–3 fold for all cell lines, p<0.05). To determine whether the results obtained following the simultaneous targeting of BCR-ABL and JAK2 in CML cell lines would extend to primary primitive CML cells and whether this combined BCR-ABL-JAK2 targeting approach could also be therapeutically effective for CML patients with inadequate responses to treatment with TKI monotherapy, we investigated CML cells obtained at the time of diagnosis from four CML patients who were classified retrospectively after the initiation of IM therapy as non-responders. To assess the effects on CFCs, CD34+ cells isolated from these samples were plated directly in methylcellose medium containing growth factors and TKIs (5μM IM and NL and 150nM DA) and BMS-911543 (100 and 300nM) alone, or in combination. The number of colonies obtained in cultures containing the respective TKIs and BMS-911543 alone was reduced from control values by about 50%. However, when TKI and BMS-911543 was present, a more significant reduction in colony formation was observed (77–86% inhibition of colony growth, p<0.05). Interestingly, combination treatment significantly inhibits BFU-E colony formation as compared with treatment with TKI alone (92–100% inhibition for the combinations vs 63–66% inhibition for the single agents, p<0.05). Most significantly, our data indicate far less toxicity on normal bone marrow CD34+ cells (n=4) as compared with CML samples treated with either single agent or combination therapy (2–3 fold from 100 to 600nM, p<0.05). The combination approach appears to be effective against treatment-naïve CML stem/progenitor cells derived from patients who were subsequently shown to be clinically resistant to IM therapy, suggesting a rational strategy for improving the clinical outcome of CML patients destined to develop IM resistance that involves the simultaneous targeting of both BCR-ABL and JAK2 in CML stem/progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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