Telomerase Inhibition with Imetelstat Eradicates β-Catenin Activated Blast Crisis Chronic Myeloid Leukemia Stem Cells

Abstract Introduction Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are generated from progenitors that have aberrantly activated self-renewal pathways thereby resulting in tyrosine kinase inhibitor (TKI) resistance. The telomerase complex, consisting of a reverse transcriptase subunit (TERT), an RNA template subunit (TERC), and a protective shelterin scaffold, transcriptionally modulates the Wnt/b-catenin self-renewal pathway. Many malignancies, including BCR-ABL TKI resistant blast crisis CML (BC CML), exhibit robust telomerase activity thereby prompting the development of imetelstat, a competitive inhibitor of telomerase enzymatic activity. Imetelstat is a covalently lipidated 13-mer oligonucleotide that binds with high affinity to the TERC subunit. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms. However, the role of imetelstat in selective self-renewing LSC inhibition in CML had not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized LSC primagraft studies to investigate the capacity of imetelstat to selectively inhibit LSC self-renewal and to determine the mechanism of action. Methods and Results Cytoscape analysis of RNA-seq data derived from FACS-purified progenitors from human blast crisis (BC; n=9) compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples revealed transcriptional upregulation of b-catenin, LEF1, TCF7L1, ABL1 and other key genes within the TERT interactome suggesting a role for TERT activation in human BC LSC generation. Human progenitor LSC-supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 mM significantly inhibited (p<0.001, ANOVA) in vitro self-renewal of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Treatment of primagraft mouse models of human BC CML with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in bone marrow progenitor LSC burden compared with mismatch treated controls (p=0.04). Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat compared with vehicle-treated samples. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human myeloid progenitors following imetelstat treatment in the TKI resistant bone marrow niche. Conclusions Niche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated LSC to imetelstat in both in vitro and in vivo humanized pre-clinical BC CML models thereby providing a strong rationale for LSC eradication trials involving imetelstat. Disclosures Huang: Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Jamieson:UC San Diego: Other: I received funding from Janssen Research & Development, LLC.

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The Role Of CD44 Isoform Expression In Niche Resident Chronic Myeloid Leukemia Stem Cell Evolution

Blood ◽

10.1182/blood.v122.21.4028.4028 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4028-4028

Author(s):

Cayla Mason ◽

Frida Holm ◽

Eva Hellqvist ◽

Christian Barrett ◽

Kelly A Frazer ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Expression Patterns ◽

Transcript Variant ◽

Bone Marrow Niche ◽

Transcript Variants ◽

Isoform Expression

Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the BCR-ABL fusion oncogene whose protein product has greatly increased ABL1 kinase activity. Although specific BCR-ABL tyrosine kinase inhibitors (TKIs) and the second generation TKIs like Nilotinib and the dual specific SCR and ABL inhibitor Dasatinib/Sprycel have dramatically improved CML therapy and significantly slowed disease progression by eradicating the bulk of CML cells in the circulation, they frequently fail to eliminate quiescent leukemic stem cells residing in the protective bone marrow niche. Leukemia stem cells (LSC) are able to drive disease relapse and may eventually contribute to the emergence of TKI resistant blast crisis (BC) CML, which is the final phase in the evolution of CML with rapid progression and short survival. CD44 is an adhesion molecule that promotes retention in the niche through adhesion to extracellular matrix components, such as hyaluronic acid and osteopontin. It plays an important role in wound healing and cell migration as well as in tumor invasion and metastasis. Through alternative mRNA splicing several CD44 isoforms exist, some of which are frequently overexpressed by cancer stem cells, including LSCs. The CD44 variant expression pattern on human blast crisis CML LSC, however, had not been elucidated. In this study we aimed to investigate the CD44 transcript variant expression of human blast crisis CML LSC. Methods and Results We performed whole transcriptome RNA sequencing of FACS sorted CML LSCs (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8), CML patients as well as the normal counterpart from cord blood (CB) (n=3) and adult peripheral blood (NPB)(n=3). A number of CD44 transcript variants were detected: v3, v4 (CD44s), v5, v6, v7, v8 plus additional variants. Earlier data suggest a total of 16 different isoforms of CD44. We found a higher overall variant gene expression of CD44 in BC compared to CP. A higher expression of CD44 transcript variant 3 and CD44 transcript variant 5 was detected in both CP and BC compared to CB and NPB. Specific CD44 transcript variant expression patterns distinguished BC progenitors from CP samples. Using splice isoform specific PCR, we were able to confirm isoform variants that were upregulated in the BC CML samples. We also compared the expression of the CD44 transcript variants in young versus old bone marrow in order to exclude that LSC-specific isoform variant expression was expressed in aged patients. We could not detect such a correlation. Conclusions These observations suggest that unique CD44 isoform expression patterns predict progression from CP to BC as well as the generation of TKI resistant LSCs and may be used as biomarkers of response to LSC targeted therapy. Disclosures: Jamieson: J&J, Roche: Research Funding; Sanofi: Membership on an entity’s Board of Directors or advisory committees.

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CD93 Is a Novel Biomarker of Leukemia Stem Cells in Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.49.49 ◽

2015 ◽

Vol 126 (23) ◽

pp. 49-49 ◽

Cited By ~ 4

Author(s):

Ross Kinstrie ◽

Gillian A. Horne ◽

Heather Morrison ◽

Hothri A. Moka ◽

Jennifer Cassels ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Fold Increase ◽

Leukemia Stem Cells ◽

Advisory Committees

Abstract The introduction of BCR-ABL tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML). However, although the majority of patients with chronic phase (CP)-CML obtain durable complete cytogenetic and major molecular responses, there is low level disease persistence postulated to be due to a population of TKI-insensitive leukemia stem cells (LSC). The aims of this study were (1) to fully characterize differences in gene expression between normal hematopoietic stem cells (HSC) and CP-CML LSC and (2) identify potential novel therapeutic targets specific to CML LSC. Lin-CD34+CD38- CD45RA-CD90+ normal HSC (n=3) and CP-CML LSC (n=6 patients at diagnosis), populations were isolated using a FACSAria and applied to Affymetrix HuGene 1.0ST arrays. The raw data (.CEL files) was imported into Partek Genomics Suite and Ingenuity Pathway Analysis software and principal component analysis and gene ontology ANOVA performed. A total of 1217 genes were significantly deregulated between normal HSC and CP-CML LSC. The most significantly deregulated genes and pathways were involved with the molecular and cellular functions of cell cycle, cell assembly and organisation, cellular movement, cell death and DNA replication, recombination and repair. These results suggested that CML LSC were less quiescent than normal HSC. Importantly, complimentary functional studies indicated that CML LSC have significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively; P<0.05). Fluorescence in situ hybridisation (FISH) demonstrated that >90% of lin- CD34+ CD38- CD45RA- CD90+ CML LSC from all patient samples were BCR-ABL positive (+). In addition to these deregulated intracellular pathways, we sought to assess if there were differences in expression of cell surface molecules that may be amenable to therapeutic manipulation. Of particular interest, our microarray studies demonstrated that CD93 was highly upregulated in CP-CML LSC (6 fold, p = 2.5x10-6). Increased CD93 expression was validated by Fluidigm digital PCR (6 fold increase, p = 0.02; n=6). Furthermore, using flow cytometry, we demonstrated significant upregulation of CD93 protein expression on lin-CD34+ CD38- CD45RA- CD90+ CML LSC from peripheral blood and bone marrow of CP-CML patients (n= 17; mean = 63.8% CD93+) compared to normal HSC from healthy peripheral blood stem cell donors (n=7; mean = 0.8% CD93+) and bone marrow donors (n=4; mean = 0.2% CD93+; p < 0.0001). FISH confirmed that 100% of lin-CD34+ CD38- CD90+ CD93+ CML cells were BCR-ABL+ in all samples assessed. CD93 (also known as C1qRp) is a C-type lectin-like domain (CTLD)-containing glycoprotein which regulates phagocytosis, with roles in cell adhesion and leukocyte migration. It is normally expressed on endothelial cells, hematopoietic precursors and mature cells including neutrophils, monocytes and platelets. Previous studies have shown CD93 to be upregulated in a proportion of AML patients (Saito et al, Sci Transl Med, 2010. 2(17): p. 17ra9). Short term (24h) in vitro exposure of lin-CD34+CD38- CD45RA- CD90+ CML LSC to TKIs (Imatinib or Dasatinib; n=3) reduced, but did not fully eliminate CD93 expression (Imatinib, 48.5% to 22.9%; Dasatinib, 47.7% to 9.2%). Importantly, following long-term TKI treatment of patients, lin-CD34+CD38-CD45RA-CD90+ cells from CP-CML patient bone marrow samples (n=2) taken in major molecular response demonstrated a small, but persistent population of CD93+ LSC which were BCR-ABL+ by FISH. Furthermore, in xenograft transplantation experiments (n = 5), after 16 weeks, CD34+CD93+ CML LSC engrafted lethally irradiated NOD/SCID/IL-2Rg-/- (NSG) mice with BCR-ABL+ cells, whereas CD34+CD93- cells from the same patient samples failed to engraft to significant levels (3.5-30 fold increase in engraftment with CD34+CD93- cells; p < 0.03). FISH confirmed that engrafted human cells were BCR-ABL+. Taken together, our results identify CD93 as a potential novel biomarker of CML LSC, which may also be helpful in assessing minimal residual disease at the LSC level. Further studies are ongoing to assess the therapeutic potential of inhibiting CD93 in CML LSC. Disclosures Copland: Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Regulation of Self-Renewal through Modulation of Let-7 Stem Cell Regulatory Family of microRNAs in Chronic Myeloid Leukemia Stem Cells

Blood ◽

10.1182/blood.v124.21.4527.4527 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4527-4527

Author(s):

Maria Anna Zipeto ◽

Qingfei Jiang ◽

Leslie A Crews ◽

Angela Court Recart ◽

Catriona HM Jamieson

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Family Members ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Qrt Pcr

Abstract Introduction Chronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSCs). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis transformation, BC LSC generation and TKI resistance. Self-renewing human BC LSCs harbor increased expression of Inflammation responsive adenosine deaminase acting on RNA (ADAR1), which can alter transcript as well as microRNA (miRNA) maturation, splicing and translation by Adenosine (A)-to-Inosine (I) editing of double stranded RNA. miRNAs are a family of small non-coding RNA molecules that regulate gene expression at a post-transcriptional level by inhibiting protein translation and/or reducing mRNA stability. Eukaryotic cells employ miRNAs in diverse biological processes including cell proliferation, differentiation, pluripotency and self-renewal. The stem cell pluripotency RNA binding protein LIN28B plays critical roles in BC transformation of CML. In this study we sought to characterize CML related-oncogenes, such as BCR-ABL, JAK2 and ADAR1, alone or in stromal co-culture in terms of their ability to regulate LSC self-renewal through modulation of let-7 /LIN28B stem cell transcriptional regulatory axis. Methods MiRNAs were extracted from purified CD34+ cells derived from CP and BC CML patient samples as well as cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human BCR-ABL, JAK2, let-7a, wild type ADAR1 and ADAR1 mutant, which lacks a functional deaminase domain. Then, 72 hours after transduction, lentivirally transduced cells were plated on irradiated SL/M2 cells. After 5 days of culture, cells were collected for RNA and microRNA extraction. Transduction efficiency and LIN28B levels were evaluated by qRT-PCR and let-7 expression was quantified by qRT-PCR using miScript primer assay. Hematopoietic Progenitor and Replating assaywere performed on lenti-let-7a-overexpressing CB cells to assess differentiation, survival and self-renewal capacity. Results Lentiviral overexpression of human BCR-ABL in CD34+ CB did not induce any significant change in let-7 family members and LIN28B expression in absence of stromal co-culture. However, stromal co-culture of BCR-ABL overexpressing CB led to the significant downregulation of members of the let-7 family as well as to upregulation of their target gene LIN28B, thus suggesting that extrinsic microenvironmental cues are necessary for modulating let-7 family levels in presence of BCR-ABL. Notably, qRT-PCR of CB transduced with JAK2 showed significant upregulation of ADAR1 in the absence of stroma, thus suggesting that JAK2 might be a mediator of inflammatory cytokine-driven ADAR1 activation. Lentiviral overexpression of both human JAK2 and ADAR1 significantly reduced the expression of let-7 family members and induced up-regulation of LIN28B. Interestingly, lentiviral overexpression of ADAR1 mutant did not induce any significant change in most let-7 family members. Finally, lentiviral overexpression of let-7a induced significant reduction in survival and self-renewal. Conclusion These finding suggest that BCR-ABL requires extrinsic signals from the niche to modulate self-renewal of BC LSCs. Conversely, lentiviral JAK2 overexpression induces activation of aberrant RNA editing and subsequent reduction of let-7 family members in the absence of the niche. Interestingly, experiments with ADAR1 mutant, suggest that ADAR1 downregulates most of the let-7 family members in a RNA–editing dependent way manner. In summary these findings suggest a novel mechanism for BC LSC generation that may have utility in prognostication and selective LSCs targeting. Disclosures Jamieson: J&J: Research Funding; Sanofi: Research Funding, Travel Support, Travel Support Other; Roche: Research Funding.

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Bone marrow niche-mediated survival of leukemia stem cells in acute myeloid leukemia: Yin and Yang

Cancer Biology and Medicine ◽

10.20892/j.issn.2095-3941.2016.0023 ◽

2016 ◽

Vol 13 (2) ◽

pp. 248-259 ◽

Cited By ~ 44

Author(s):

Hong-Sheng Zhou ◽

Bing Z. Carter ◽

Michael Andreeff ◽

Bing Z. Carter ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemia Stem Cells ◽

Bone Marrow Niche ◽

Yin And Yang ◽

Acute Myeloid

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Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

Stem Cells International ◽

10.1155/2020/8885154 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Huihong Zeng ◽

Jiaoqi Cheng ◽

Ying Fan ◽

Yingying Luan ◽

Juan Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Bone Marrow Niche ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

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Targeting Chronic Myeloid Leukemia (CML) Stem Cells by BMS-214662 in Mice.

Blood ◽

10.1182/blood.v106.11.2861.2861 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2861-2861

Author(s):

Cong Peng ◽

Yiguo Hu ◽

Francis Y. Lee ◽

Shaoguang Li

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Cancer Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Philadelphia Chromosome ◽

Inhibitory Effect ◽

Leukemia Stem Cells ◽

Marrow Cells

Abstract The BCR-ABL inhibitor imatinib mesylate is the current approved treatment for Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML). While this agent is effective in the chronic phase of CML, it is less effective in advanced disease (acelerated phase or blast crisis), and resistance to imatinib is an issue at all stages of disease, particularly advanced. Resistance is mediated primarily by BCR-ABL mutations, although other mechanisms have also been implicated. Another key issue with imatinib therapy is that molecular remission in imatinib-treated CML patients is difficult to achieve, leaving patients at risk of relapse. We have previously observed that imatinib significantly prolongs survival of CML mice, but is not curative (Hu et al, Nature Genetics36[5]:453–461, 2004). We hypothesize that this can be attributed to the inability of imatinib to completely kill CML stem cells. We identified that BCR-ABL-expressing Lin-c-KIT+Sca-1+ bone marrow cells are CML stem cells in mice. We tested whether BMS-214662 (which has been shown to have an inhibitory effect on growth of non-proliferating cancer cells) (Lee et al, Proceedings of the AACR42:260s, 2001) reduces leukemia stem cell populations in CML mice. Donor bone marrow cells from C57BL/6 mice were transduced with P210BCR-ABL-IRES-GFP retrovirus, followed by transplantation into lethally irradiated C57BL/6 recipient mice. Eight days after transplantation, BMS-214662 was given orally once a day at a dose of 300 mg/kg for 7 days. Bone marrow cells from the treated CML mice were then analyzed by FACS for CML stem cells (GFP+Lin-c-Kit+Sca-1+). CML mice treated with placebo, dasatinib (a novel, oral, multi-targeted kinase inhibitor that targets BCR-ABL and SRC family kinases) 10 mg/kg, twice daily (BID), BMS-214662, or dasatinib 10 mg/kg BID in combination with BMS-214662. Numbers of leukemia stem cells per bone were significantly lower in mice treated with BMS-214662 alone, dasatinib alone, or both BMS-214662 and dasatinib, compared with placebo-treated mice. Among different treatments, the combination of BMS-214662 and dasatinib had the strongest inhibitory effect on CML stem cells. Inhibition of the leukemia stem cells by dasatinib could be due to its inhibitory effect on BCR-ABL or SRC kinases, whereas BMS-214662 must function through other mechanisms. BMS-214662 is also a farnesyl transferase inhibitor (FTI), which reduces Ras activation. However, our control experiment showed that other FTIs did not inhibit proliferation of non-proliferating cancer cells (data not shown). This suggests that BMS-214662 inhibits CML stem cells through unknown mechanisms. In summary, BMS-214662 is a potent inhibitor of CML stem cells, and combinatorial use of BMS-214662 and dasatinib may provide more durable responses, and potentially a curative therapy for CML patients. Given the proven activity of dasatinib against a spectrum of imatinib-resistant BCR-ABL mutations (O’Hare, et al. Cancer Res65[11]:4500–5, 2005; Shah et al, Science, 305:399, 2004), and the apparent activity of dasatinib against stem cells in vivo shown here, this combination could potentially suppress the emergence of resistance, further adding to the durability of response.

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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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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 ◽

10.1182/blood.v116.21.204.204 ◽

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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BCL6 Is Required for the Maintenance of Leukemia-Initiating Cells In Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.202.202 ◽

2010 ◽

Vol 116 (21) ◽

pp. 202-202 ◽

Cited By ~ 1

Author(s):

Christian Hurtz ◽

Cihangir Duy ◽

Leandro Cerchietti ◽

Katerina Chatzi ◽

Eugene Park ◽

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Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Side Population ◽

Blast Crisis ◽

Scid Mice ◽

Leukemia Stem Cells ◽

Leukemia Stem Cell ◽

Bcl6 Expression ◽

Tki Treatment

Abstract Abstract 202 Background: Chronic myeloid leukemia (CML) can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia stem cells in CML. Relapse from leukemia stem cells in CML often results in TKI-resistant blast crisis, which is fatal within months. Approach: Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in chronic myeloid leukemia (CML). The mechanism of FoxO3A-dependent maintenance of leukemia stem cells remained unclear. Here we identified the BCL6 protooncogene downstream of FoxO3A as a critical effector molecule of self-renewal signaling in CML-initiating cells. BCL6 is known as a proto-oncogene in Diffuse Large B cell Lymphoma (DLBCL), where it functions as transcriptional repressor of p53. BCL6-Null CML cells fail to initiate leukemia: Studying gene expression changes of CML cells in 6 patients before and after treatment with Imatinib, we found that BCL6 mRNA levels were increased by >15-fold in response to Imatinib-treatment. Studying CD34+ CD38- CML cells from leukapheresis samples of two patients and CML cell lines, we found that overnight incubation with Imatinib resulted in a >12-fold increase of BCL6 expression at the mRNA and protein level. Previous studies showed that FoxO factors are required for transcriptional activation of BCL6 and FoxO3A was recently identified as a critical factor of leukemia stem cell maintenance in CML. Here we showed that inducible activation of FoxO3A indeed leads to de novo expression of BCL6 in human CML cells. We next tested the functional significance of BCL6 expression in CML cells in a genetic experiment. To this end, we used a classical mouse model for CML-like leukemia and transformed Lin− Sca-1+ c-kit+ (LSK) cells from BCL6+/+ and BCL6-Null mice with BCR-ABL1. While CML transformation efficiency was similar for BCL6+/+ and BCL6-Null LSK cells, the LSK phenotype was rapidly lost in BCL6-Null CML cells. To elucidate the mechanism of progressive loss of LSK cells in BCL6-Null CML, we performed a systematic analysis of gene expression changes in BCL6+/+ and BCL6-Null CML cells: The ABCG2 transporter, which is required for the side population (SP+) phenotype in LSK cells was reduced by >7-fold in the absence of BCL6. SP+ LSK cells represent a highly drug resistant CML subpopulation with leukemia-initiation capacity. In addition, BCL6-Null CML cells express excessively high levels of p53 at the protein level. A genome-wide mapping approach of BCL6-DNA interactions using ChIP-seq showed that BCL6 strongly binds to and represses the p53 promoter. Importantly, BCL6 expression represents a critical requirement for CML cells to form colonies in semisolid agar. Compared to BCL6+/+ CML cells (>400 colonies/10,000 cells), colony formation by BCL6-Null CML cells was reduced by ≂f100-fold (<5 colonies). After an initially successful engraftment of BCL6-Null CML cells in NOD/SCID mice (as visualized by luciferase bioimaging), BCL6-Null CML cells failed to initiate leukemia, whereas NOD/SCID mice injected with BCL6+/+ CML cells succumbed to the disease. Targeted inhibition of BCL6 for leukemia stem cell eradication in CML: A retro-inverso BCL6 peptide inhibitor (RI-BPI) was recently developed for targeted therapy of DLBCL. Therefore, we tested the potential therapeutic usefulness of RI-BPI for eradication of BCL6-dependent leukemia-initiating cells. Human CML cells were incubated overnight in the presence of 5 μ mol/l RI-BPI or vehicle control. While Hoechst 33342 staining revealed a distinct side population of ≂f2.8% CML cells, overnight incubation with RI-BPI reduced the frequency of ABCG2+ side population cells by more than 30-fold. Likewise, RI-BPI incubation decreased colony formation by >5-fold in human CML cells. Importantly, RI-BPI treatment of CML cells resulted in significantly prolonged overall survival of xenografted NOD/SCID mice and decreased penetrance of leukemia (10 mice/group; p=0.032), Conclusion: Pharmacological inhibition of BCL6 represents a powerful strategy to eradicate leukemia-initiating cells in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation. Disclosures: Hochhaus: Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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