Chronic Myeloid Leukemia Stem Cells

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.

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A Bio-Integrative Approach Identifies an Inflammatory Signature in Chronic Myeloid Leukemia (CML) Stem Cells That Is Highly Perturbed in CML Blast Crisis and Involves REL transcription Factor

Blood ◽

10.1182/blood.v126.23.4017.4017 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4017-4017

Author(s):

Christophe Desterke ◽

Ludovic Marie-Sainte ◽

Amine Sbitti ◽

Ali Naama ◽

Annelise Bennaceur-Griscelli ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Integrative Approach ◽

Hematopoietic Stem ◽

Gene Set

Abstract Chronic myeloid leukemia is a clonal myeloproliferative neoplasm defined by the presence of BCR-ABL fusion gene. This oncogenic event occurs in a hematopoietic stem cell (HSC) involved in CML initiation, maintenance, relapse and progression. Several evidences suggest that inflammatory pathways may participate to the pathophysiology of CML as well as disease progression to blast crisis. It has been shown that NFKB/REL pathway is constitutively activated both in BCR-ABL positive leukemic cell lines as well as in primary blast cells from CML-BC patients. More recent works identified IL6 as key cytokine acting on CML multipotent progenitors and their normal bystander counterpart to favor their differentiation toward the myeloid lineage. In addition, high levels of autocrine TNFα secretion by quiescent CML stem/progenitor cells activate NFKB pathway and promote their survival. Although all of these observations are linked to inflammatory processes, a focused analysis of inflammatory pathways in primary CML stem cells has not been performed so far. In the present study we undertook a text-mining strategy using pubmed e-querying to generate an exhaustive set of genes linked to inflammation. Then we integrated transcriptome analysis of highly purified CML stem cells to evaluate the contribution of these genes in CML development and progression. Methods : We queried 6 key words (Inflammation, macrophages, inflammatory response, chemokines, leukocytes and interleukins) that returned a total of 332000 hits in Pubmed. A raw gene set of 918 genes was found significantly associated (p<0.05) with these hits. Using R-package, we applied a false discovery rate correction that decreased the set to 588 relevant genes. The expression level of this gene set was then analyzed in previously reported microarray data (GEO accession: GSE47927) of highly purified normal cord blood CD34+CD38-CD90+ HSCs (CB; n=3), chronic phase (CP; n= 6), accelerated phase (AP; n =4) and Blast crisis (BC; n=2) CML cells. Results: Among the 588 genes related to inflammation we found 70 genes differentially expressed between the four groups (normal, CP, AP and BC, p<0.01; ANOVA test). Enrichment analysis confirmed 29 up regulated genes (NES = 2.12; p<0.0001) among which IL-6, PARP1, IL1R2, IRF5, IRF8, IL20. 39 genes such as STAT3, STAT4, CD47, CXCR4 IL-11, IL15, TLR-1, were down-regulated in CML CD34+CD38-CD90+ (all phases) as compared with normal HSCs (NES = -2,58; p<0.0001). Using principal component analysis on the 70 inflammatory deregulated genes we identified 10 genes such as IRAK1, IL1R2, VEGF and ESAM that discriminate "all phase" CML samples from normal HSCs (Dim 2 = 22.7%). Another inflammatory gene subset (n=26 genes) comprising IL6, REL, CXCR4, CXCL2, IL11, TLR1, IL1R2, PPARA highly separated CML stem cells according to the disease phase. The later gene set highly separates CP and AP-CML stem cells from BC-CML stem cell (Dim 1 = 50.3%). We next performed a random forest analysis with machine learning (1000 trees) and found that the inflammatory transcript level that best predicted CML phase was REL transcription factor. The expression of 413 genes were found positively correlated with REL expression in CP, AP and BC-CML CD34+CD38-CD90+ cells (r>0.75 and p-value <0.001). A search using JASPAR and TRANSFAC database identified a significant enrichment of NFKB1 and RELA binding motif in the promoter regions of these 413 genes (p<0.00001) among which several regulatory factors of hematopoietic stem cell biology. Conclusion : Using a bio-integrative approach we identified a specific inflammatory signature in CD34+CD38-CD90+ CML stem cells. This inflammatory network is highly altered in blast crisis suggesting its contribution to disease evolution. We identified REL overexpression as a good predictor for disease progression to blast crisis and found NFKB1and RELA (p=3.2x10-13) as the best REL target candidates. RELA/NFKB1 was previously shown to be constitutively activated in CML and Ph+ ALL and this analysis suggests that this may also take place in the most primitive subset of CML cells although REL may be the main partner of NFKB in CML stem cells. These results which are currently validated using functional assays, could lead to identification of novel therapeutic strategies. Disclosures Turhan: Bristol Myers Squibb: Consultancy; Novartis: 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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Centrosome Aberrations in Chronic Myeloid Leukemia (CML) Are Correlated with Disease Progression and Chromosome Instability.

Blood ◽

10.1182/blood.v104.11.2949.2949 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2949-2949

Author(s):

Michelle Giehl ◽

Alice Fabarius ◽

Chun Zheng ◽

Oliver Frank ◽

Andreas Hochhaus ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Chromosome Instability ◽

Chronic Phase ◽

Normal Karyotype ◽

Benign Tumors ◽

Preneoplastic Lesions ◽

Cd34 Cells

Abstract Purpose: Numerical and structural centrosome abnormalities are hallmarks of a variety of cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. These phenomena already occur in preneoplastic lesions like oral leukoplakia, early cervical neoplasias, and small benign tumors of colon and breast. Moreover, deviations from normal karyotype seem to increase as tumors enlarge and become malignant. Genetic instability is a common feature in chronic myeloid leukemia (CML). We sought to establish a relationship between centrosome abnormalities and cytogenetic aberrations in CD34+ cells from CML patients at diagnosis (chronic phase - CP) and in blast crisis (BC). Methods: Diagnosis of CML was established by hematologic, cytogenetic and molecular parameters. Treatment was performed according to the protocols of the German CML study group (www.kompetenznetz-leukaemie.de). CD34+ cells from ten umbilical cord blood specimens served as negative controls. Centrosome number and morphology were analyzed by immunofluorescence microscopy. In brief, CD34+ cells from ficollized peripheral blood samples were concentrated by magnetic cell sorting (MACS) and cytospun onto coated slides. After methanol fixation cells were incubated with antibodies directed to centrosomal proteins Pericentrin and gamma-Tubulin. Antibody-antigen complexes were stained by incubation with FITC- and Cy3-conjugated secondary antibodies. Results: CML CP samples tested at initial diagnosis (n=20) already displayed numerical and structural centrosome aberrations (30.0% +/−2.3) as compared with corresponding normal control cells (n=10) (2.3% +/−1.1). In BC samples (n=10) an increase of centrosome aberrations was observed (58.0% +/−2.0). Conclusion: The findings suggest that centrosome defects in CML occur early and are already present at primary diagnosis. Centrosome defects may contribute to disease progression by generation of further chromosome instability leading to accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing normal tumor suppressor genes and thus accelerating complex genomic changes associated with CML BC.

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Cytogenetic Analysis of 1863 Chineses Patients with Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.4888.4888 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4888-4888

Author(s):

Qitian Mu ◽

Qiuling Ma ◽

Yungui Wang ◽

Xiangmin Tong ◽

Zhimei Chen ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Chronic Phase ◽

Newly Diagnosed ◽

Monocytic Differentiation ◽

Granulocytic Differentiation ◽

Monosomy 7

Abstract Abstract 4888 Background: Cytogenetic analyses of chronic myeloid leukemia (CML) have been performed previously in a large number of reports, but systematical research based on large sample sizes is seldom available. In order to further elucidate the cytogenetic nature of CML, we analyzed retrospectively the cytogenetic profiles of 1863 Ph/BCR-ABL-positive CML patients from a research center in China. Results: Of 1266 newly diagnosed CML patients, the median age was 41 years, which is younger than the median age of diagnosis in western populations. The incidence of additional chromosome abnormalities(ACAs) was 3.1% in newly-diagnosed chronic phase(CP), 9.1% in CP after therapy, 35.4% in accelerated phase(AP) and 52.9% in blast phase(BP), reflecting cytogenetic evolution with CML progression. 5.3% patients harbored a variant Ph translocation. A higher prevalence of ACAs was observed in variant Ph translocations than in classical t(9;22) in the disease progression, especially in BP(88.2% vs. 50%, p=0.002). Moreover, a hyperdiploid karyotype and trisomy 8 were closely correlated with myeloid blast crisis(BC) while a hypodiploid karyotype and monosomy 7 were associated with lymphoid-BC. Among subsets of myeloid-BC, compared with myeloid-BC with granulocytic differentiation or monocytic differentiation, myeloid-BC with minimal differentiation had higher ACAs rate (80% vs.46.8%, p=0.009 and 80% vs. 42.9%, p=0.006). Conclusion: CML tends to afflict younger population in China. In the disease progression, the incident of ACAs was higher in variant Ph translocations than in classical t(9;22). Among subsets of myeloid-BC, myeloid with minimal differentiation had distinct cytogenetic features. Disclosures: No relevant conflicts of interest to declare.

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B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Specific Copy Number Alterations Are Unique For Progressive Pediatric Chronic Myeloid Leukemia (CML): A Large Cohort Study

Blood ◽

10.1182/blood.v122.21.2715.2715 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2715-2715

Author(s):

Naomi E van der Sligte ◽

Manuela Krumbholz ◽

Agata Pastorczak ◽

Blanca Scheijen ◽

Josephine T. Tauer ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

B Cell ◽

Disease Progression ◽

Copy Number ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Copy Number Alterations ◽

Cell Precursor ◽

Predict Disease Progression

Abstract Chronic myeloid leukemia (CML) is a rare malignancy in children and is mostly diagnosed in the chronic phase (CP). In adults, the five-year overall survival rate is 89% for patients on Imatinib and disease progression occurs in 1-3% per year (Druker 2006). Once a blast crisis (BC) has occurred, treatment options are limited with a median survival of only a few months (Cortes 2008). Therefore, early recognition of patients at risk for developing a BC is desirable. Besides the translocation t(9;22)(q34;q11), IKZF1, PAX5, and CDKN2A deletions have been reported in CML lymphoid blast crisis (LyBC) of both adult and pediatric patients (Mullighan 2008, Alpár 2012). The aim of this study was to investigate the presence of IKZF1 deletions and other copy number alterations (CNAs) by MLPA analysis in a large cohort of pediatric CML patients at time of diagnosis in order to determine whether CNAs commonly found in pediatric ALL might predict disease progression and / or treatment response. Between October 1991 and October 2012 a total of 86 children with newly diagnosed CML were included. The median follow up was 31 months. Among the 86 patients, 82 patients were diagnosed in CP, 2 patients in accelerated phase (AP), and 2 patients in LyBC. Six patients experienced progression to a BC respectively a myeloid blast crisis (MyBC) (N=2) and LyBC (N=4). At time of diagnosis, an IKZF1 deletion was detected in one patient diagnosed with CML-AP (Table A, patient no 58). IKZF1 and EBF1 deletions were detected in one patient diagnosed with CML-LyBC (Table A, patient no 22). No CNAs were detected in the 82 patients diagnosed with CML-CP. At time of disease progression, new CNAs were detected at time of the LyBC (Table A, patient no 62, 64, and 67). Due to the absence of material no CNAs could be detected in both patients experiencing a MyBC. In conclusion, we were able to detect CNAs in progressive CML disease (CML-AP and CML-LyBC) and not in the samples at the time of chronic phase in this large pediatric cohort of CML patients. Therefore, the investigated CNAs could not be used to predict disease progression at time of diagnosis. The CNAs detected in patients with progressive CML were similar to specific CNAs detected in pediatric B-cell precursor ALL, indicating a similar disease development (Kuiper 2010). Additionally, our results are in accordance with existing literature, suggesting that mechanisms of disease progression in pediatric and adult CML might be similar (Brazma, 2007). Disclosures: No relevant conflicts of interest to declare.

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Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Chronic Myeloid Leukemia in Second Chronic Phase Attained by Imatinib after Onset of Blast Crisis.

Blood ◽

10.1182/blood.v110.11.4566.4566 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4566-4566

Author(s):

Ying Wang ◽

De Pei Wu ◽

Aing-Ning Sun ◽

Zheng-ming Jin ◽

Miao Miao

Keyword(s):

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Hematopoietic Stem ◽

Allogeneic Hsct

Abstract The prognosis for patients with chronic myeloid leukemia (CML) in blast crisis (BC) remains dismal even with the availability of the BCR-ABL tyrosine kinase inhibitor imatinib, since it only offers short-term benefit in most cases. Allogeneic hematopoietic stem cell transplantation (HSCT) seems to be a viable option for BC-CML patients who attained remission. We treated 10 patients (9 males, 1 female) with ablative allogeneic HSCT, who achieved second chronic phase (CP) by the use of imatinib after onset of BC between October 2003 and August 2006. Median patient age was 32 years (range, 17–46). Imatinib was given orally at daily doses ranging from 600 to 800mg according to patients tolerance for at least 2 months (range, 2–5) prior to HSCT Among them, 4 patients received HSCT from human leukocyte antigen mismatched haplo-identical family donors, the others underwent a transplant from HLA matched related (n=5) or unrelated (n=1) donors. At the time of transplantation, 5 patients were in complete hematologic response with 3 patients achieved a cytogenetic response, 5 patients were in partial hematologic response. After a median follow-up of 26 months (range, 10–44), 6 (60%) out of the 10 patients were alive with mean Karnofsky score reaching 80. Among them, 5 patients achieved a molecular remission. 1 patient died in relapse 4 months after transplantation, the others died of severe acute graft-versus-host disease and associated infections. No unusual organ toxicities and engraftment difficulties were observed. Extensive chronic GVHD developed in 3 of 6 patients who could be evaluated. Patients transplanted with haplo-identical donors had a high treatment-related modality, 3 out of 4 patients died. These results suggest that allogeneic HSCT may represent a feasible treatment for patients with CML in second CP attained by imatinib after onset of BC especially when a suitable donor is available.

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Increase of bcr-abl chimeric mRNA expression in tumor cells of patients with chronic myeloid leukemia precedes disease progression

Blood ◽

10.1182/blood.v86.6.2371.bloodjournal8662371 ◽

1995 ◽

Vol 86 (6) ◽

pp. 2371-2378 ◽

Cited By ~ 114

Author(s):

A Gaiger ◽

T Henn ◽

E Horth ◽

K Geissler ◽

G Mitterbauer ◽

...

Keyword(s):

Steady State ◽

Chronic Myeloid Leukemia ◽

Mrna Expression ◽

Disease Progression ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Blast Crisis ◽

Chronic Phase ◽

Leukemic Cells ◽

Phenotypic Transformation

The translocation t(9;22) in chronic myeloid leukemia (CML) generates a bcr-abl fusion gene that codes for an aberrant chimeric mRNA. Cell lines established from CML patients in blast crisis show higher expression of this aberrant bcr-abl transcript than cells from patients in chronic phase of the disease. This observation provided the stimulus to investigate whether increased expression of the aberrant bcr-abl fusion transcript is critical to the progression of CML from chronic phase to blast crisis. We have monitored the bcr-abl mRNA expression in 25 patients by serial quantitative polymerase chain reaction analyses during a follow-up period of 12 to 156 months after diagnosis, with a median observation time of 28 months. In all patients who have shown disease progression to accelerated phase (n = 4) or blast crisis (n = 7), an increase in bcr-abl mRNA expression was detected up to 16 months before laboratory or clinical parameters showed phenotypic transformation of the malignant clone. To investigate whether the elevated levels of bcr-abl mRNA reflected an increase in the proportion of leukemic cells in the samples analyzed or primarily enhanced transcriptional activity of the bcr-abl fusion gene, we performed quantitative analyses of the bcr-abl gene at the DNA level and of the Ph chromosome at the cytogenetic level and compared these data with steady-state bcr-abl mRNA levels. We show that increased levels of the bcr-abl transcript did not reflect increased proportions of leukemic cells but elevated steady-state levels of the chimeric mRNA in the malignant cells before disease progression. Therefore, our data strongly suggest that an increase of the chimeric mRNA expression in the leukemic cells precedes the phenotypic transformation of the malignant clone.

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

Blood ◽

10.1182/blood.v128.22.3065.3065 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3065-3065

Author(s):

Wenxue Ma ◽

Cayla N Mason ◽

Ping Chen ◽

Nathaniel Delos Santos ◽

Jiang Qingfei ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Leukemia Stem Cells ◽

Bone Marrow Niche ◽

Rna Seq ◽

Self Renewal

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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Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia

Biology ◽

10.3390/biology10111182 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1182

Author(s):

Zafar Iqbal ◽

Muhammad Absar ◽

Tanveer Akhtar ◽

Aamer Aleem ◽

Abid Jameel ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Sanger Sequencing ◽

Structural Changes ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Chronic Phase ◽

Genomic Analysis

Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

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Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia

10.20944/preprints202108.0498.v1 ◽

2021 ◽

Author(s):

Zafar Iqbal ◽

Muhammad Absar ◽

Tanveer Akhtar ◽

Aamer Aleem ◽

Abid Jameel ◽

...

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Sanger Sequencing ◽

Structural Changes ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Chronic Phase ◽

Genomic Analysis

Background: Chronic Myeloid Leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(22;9) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKI) have changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find out novel biomarkers of CML progression by employing whole exome sequencing (WES).Materials and Methods: WES of accelerated phase (AP-) and blast crisis (BC-) CML patients was carried out, with chronic phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing was carried out using Illumina platforms. Statistical analysis was carried out using [SAS/STAT] software version 9.4 and R package employed to find mutations shared exclusively by all AP-/BC-CML. Confirmation of mutations was carried out using Sanger sequencing and protein structure modelling using I-Tasser followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modelling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in human. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in biology and progression of CML.

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