Reversion to an Embryonic Alternative Splicing Program Enhances Leukemia Stem Cell Self-Renewal

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1227-1227
Author(s):  
Frida Linnea Holm ◽  
Eva Hellqvist ◽  
Cayla N Mason ◽  
Shawn Ali ◽  
Nathaniel Delos Santos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Alternative Splicing ◽  
Blast Crisis ◽  
Embryonic Stem ◽  
Chronic Phase ◽  
Cell Reprogramming ◽  
Rna Seq ◽  
Self Renewal ◽  
Splice Isoform

Abstract Background Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established. Methods Whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013). Results We performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance. Conclusion In summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. Disclosures Jamieson: Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Cycling Toward Leukemia Stem Cell Elimination Wtih a Selective Sonic Hedgehog Antagonist,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3776-3776 ◽  
Author(s):  
Alice Y Shih ◽  
Annelie Schairer ◽  
Christian L Barrett ◽  
Ifat Geron ◽  
Angela C Court Recart ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Rna Sequencing ◽  
Sonic Hedgehog ◽  
Blast Crisis ◽  
Self Renewal ◽  
Shh Signaling ◽  
Splice Isoform ◽  
Cell Cycle Regulatory Genes

Abstract Abstract 3776 Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse. Disclosures: Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.

Chronic Myeloid Leukemia Stem Cells

Hematology ◽  
2008 ◽  
Vol 2008 (1) ◽  
pp. 436-442 ◽  
Author(s):  
Catriona H. Jamieson
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Targeted Therapy ◽  
Chronic Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Progenitor Cell ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Self Renewal

Abstract Chronic myeloid leukemia (CML) is typified by robust marrow and extramedullary myeloid cell production. In the absence of therapy or sometimes despite it, CML has a propensity to progress from a relatively well tolerated chronic phase to an almost uniformly fatal blast crisis phase. The discovery of the Philadelphia chromosome followed by identification of its BCR-ABL fusion gene product and the resultant constitutively active P210 BCR-ABL tyrosine kinase, prompted the unraveling of the molecular pathogenesis of CML. Ground-breaking research demonstrating that BCR-ABL was necessary and sufficient to initiate chronic phase CML provided the rationale for targeted therapy. However, regardless of greatly reduced mortality rates with BCR-ABL targeted therapy, most patients harbor quiescent CML stem cells that may be a reservoir for disease progression to blast crisis. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, only recently have the HSC and progenitor cell–specific effects of the molecular mutations that drive CML been investigated. This has provided the impetus for investigating the genetic and epigenetic events governing HSC and progenitor cell resistance to therapy and their role in disease progression. Accumulating evidence suggests that the acquired BCR-ABL mutation initiates chronic phase CML and results in aberrant stem cell differentiation and survival. This eventually leads to the production of an expanded progenitor population that aberrantly acquires self-renewal capacity resulting in leukemia stem cell (LSC) generation and blast crisis transformation. Therapeutic recalcitrance of blast crisis CML provides the rationale for targeting the molecular pathways that drive aberrant progenitor differentiation, survival and self-renewal earlier in disease before LSC predominate.

scholarly journals Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal

2015 ◽  
Vol 112 (50) ◽  
pp. 15444-15449 ◽  
Author(s):  
Frida Holm ◽  
Eva Hellqvist ◽  
Cayla N. Mason ◽  
Shawn A. Ali ◽  
Nathaniel Delos-Santos ◽  
...  
Keyword(s):  
Stem Cell ◽  
Alternative Splicing ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Combined Treatment ◽  
Embryonic Stem ◽  
Chronic Phase ◽  
Dependent Manner ◽  
Splice Isoform

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

scholarly journals Malignant Reprogramming of Progenitors into Leukemia Stem Cells Is Enhanced By Upregulation of CD44 Transcript Variant 3 in Malignant Microenvironments

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 511-511
Author(s):  
Frida L Holm ◽  
Eva Hellqvist ◽  
Cayla N Mason ◽  
Christian L Barrett ◽  
Shawn Ali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Expression Analysis ◽  
Splice Variant ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Transcript Variant ◽  
Rna Seq ◽  
Cd44 Variant

Abstract Introduction Malignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche. Methods and Results CD44 Isoform Expression Analysis To investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34+CD38+Lin-) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency. Lentiviral CD44 Variant 3 Overexpression To directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency. Targeted Inhibition of CD44 variant 3 Expressing LSC Humanized RAG2-/-gc-/-mice engrafted with CD34+ BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression. Conclusions Upregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs)

2017 ◽  
Vol 44 (4) ◽  
pp. 1435-1444 ◽  
Author(s):  
Hong-Xi Zhao ◽  
Feng Jiang ◽  
Ya-Jing Zhu ◽  
Li Wang ◽  
Ke Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Human Embryonic Stem Cells ◽  
Immune Tolerance ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Immunological Tolerance ◽  
Self Renewal ◽  
Hesc Lines

Background: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. Methods: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. Results: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. Conclusions: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

scholarly journals Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jordi Solana ◽  
Manuel Irimia ◽  
Salah Ayoub ◽  
Marta Rodriguez Orejuela ◽  
Vera Zywitza ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Alternative Splicing ◽  
Cell Biology ◽  
Embryonic Stem ◽  
In Vivo Model ◽  
Stem Cell Regulation ◽  
Specific Alternative ◽  
Functional Antagonism

In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans.

scholarly journals Alternative Splicing in Self-Renewal of Embryonic Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Clara Y. Cheong ◽  
Thomas Lufkin
Keyword(s):  
Stem Cells ◽  
Alternative Splicing ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Cell Biology ◽  
Genomic Sequence ◽  
Splice Variants ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Additional Layer

Much of embryonic stem cell biology has focused on transcriptional expression and regulation of genes that could mediate its unique potential in self-renewal or pluripotency. In alignment with our present understanding on the genetic, protein, and epigenetic factors that may direct cell fate, we present a short overview of the often overlooked contribution of alternative splice variants to regulatory diversity. Progressing beyond the limitations of a fixed genomic sequence, alternative splicing offers an additional layer of complexity to produce protein variants that may differ in function and localization that can direct embryonic stem cells to specific differentiation pathways. In light of the number of variants that can be produced at key ES cell genes alone, it is challenging to consider how much more multifaceted transcriptional regulation truly is, and if this can be captured more fully in future works.

Stromal Cell-Derived Secreted Factors Contribute to the Innate Imatinib Resistance of Leukemia Stem Cells In a Genetically Defined Murine Model of Chronic Myeloid Leukemia Blast Crisis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 204-204
Author(s):  
C. Ronald Geyer ◽  
Michael Szeto ◽  
Ashton Craven ◽  
Marciano D. Reis ◽  
David P. Sheridan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cell ◽  
Blast Crisis ◽  
Conditioned Media ◽  
Imatinib Resistance ◽  
Self Renewal ◽  
Soluble Factors

Abstract Abstract 204 Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures: No relevant conflicts of interest to declare.

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