Transcriptome of Expressed Genes in CD34+ and CD66b+ Chronic Myeloid Leukemia Cells and Its Potential Role in the Transport of Kinase Inhibitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4426-4426
Author(s):  
Caroline Fátima Aquino Moreira-Nnes ◽  
Ana Cristina Simões Beltrão ◽  
Tereza Cristina Brito Azevedo ◽  
Larissa Tatiana Valente Martins Francês ◽  
Rodrigo Guarischi Mattos Amaral Sousa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Leukemic Cells ◽  
Cancer Resistance ◽  
Optimal Response ◽  
Oncology Research ◽  
Cd34 Cells ◽  
Long Term Treatment

Abstract Abstract 4426 Background. Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by Phildaelphia chromosome and by formation of BCR-ABL fusion. Some studies have shown that residual cells are part of the leukemic cells undifferentiated compartment. In 2005, Michor et al. (Nature 435: 1267–1270), through mathematical model, concluded that imatinib efficiently reduces the differentiated leukemic cells population, but it has not the same effect on the cell population that drives this disease, the CD34+ leukemic stem cells, which can be kept alive during the treatment. However, Mahon et al. (Lancet Oncol 11: 1029–35, 2010) described a cohort in which patients remained disease free for 18 months after discontinued treatment. This finding is an indication that the leukemia stem cells (LSC) are not totally insensitive to kinase inhibitors (KIs). Purpose. Identify the expressed genes in CD34+ and CD66b+ cells as candidates for KIs transport. Methods. Samples of bone marrow (BM) and peripheral blood (PB) were obtained from five patients with CML treated with imatinib in optimal response in according to European LeukemiaNet criteria. Cells Isolation and RNA extraction. CD34+ cells were isolated from BM of five patients with CML. Mature CD66b+ PB cells were isolated from the same patients. SOLiD sequencing. cDNA was sequenced according to the manufacturer's protocols for the SOLiD. Transcriptome. Two libraries were constructed for this purpose and approximately 120 millions of beads were deposited on a half slide for each library and sequenced using the Opti Fragment Library Sequencing kit-Master Mix 50 on a SOLiD machine (Ver 3+). To characterize the genes showing differential regulation, we analyzed the Gene Ontology (GO) annotation associated with transporters genes exhibiting a greater than 2-fold difference in expression by RNA-seq. Analysis using the functional annotation clustering feature of DAVID. Results. We have sequenced 14.133 genes in CD34+ pool cells and 3.379 genes in CD66b+ pool cells, with 2.883 genes expressed in both. Of these, 1.201 genes from membrane transport were functionally annotated, and we have found 560 genes expressed exclusively in CD34+, and 99 genes in CD66b+, and 542 genes in both, as shown below. Regarding imatinib transportation, two major classes of transporters are widely recognized for its importance in drug influx and efflux inside the cell, the ATP-biding cassette transporters (ABC family) and Solute Carrier family (SLC family). Studies have demonstrated that the organic cation transporter 1 (OCT-1, also known as SLC22A1) is the major active influx transporter for imatinib in CML cells, in our study we find no evidence of OCT-1 in any of our samples, which may indicate that this is not the channel for the influx of drugs into cells. Within ABC family is already recognized the important function of at least three genes in multidrug-resistance cancer through the mechanism of drug efflux through the membrane, they are: ABCB1 (MDR1); ABCG2 (Breast Cancer Resistance Protein – BCRP) e ABCC1 (MRP1). These genes were found exclusively in the CD34+ lineage of CML patients, reinforcing the theory that HSC are resistant to treatment with imatinib, through the expression of efflux channels. Conclusion. The efflux channel genes exclusively expressed in CD34+ cells represents a major barrier to maintaining optimal response to KIs in long-term treatment of CML patients. Those genes should be investigated to achieve the development of drugs with potential to block the efflux channels and improve outcome for cancer patients. Disclosures: Lemos: Novartis Oncology: Research Funding.

scholarly journals Druggable Biochemical Pathways and Potential Therapeutic Alternatives to Target Leukemic Stem Cells and Eliminate the Residual Disease in Chronic Myeloid Leukemia

2019 ◽  
Vol 20 (22) ◽  
pp. 5616 ◽  
Author(s):  
Fabien Muselli ◽  
Jean-François Peyron ◽  
Didier Mary
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Hematopoietic Stem

Chronic Myeloid Leukemia (CML) is a disease arising in stem cells expressing the BCR-ABL oncogenic tyrosine kinase that transforms one Hematopoietic stem/progenitor Cell into a Leukemic Stem Cell (LSC) at the origin of differentiated and proliferating leukemic cells in the bone marrow (BM). CML-LSCs are recognized as being responsible for resistances and relapses that occur despite the advent of BCR-ABL-targeting therapies with Tyrosine Kinase Inhibitors (TKIs). LSCs share a lot of functional properties with Hematopoietic Stem Cells (HSCs) although some phenotypical and functional differences have been described during the last two decades. Subverted mechanisms affecting epigenetic processes, apoptosis, autophagy and more recently metabolism and immunology in the bone marrow microenvironment (BMM) have been reported. The aim of this review is to bring together the modifications and molecular mechanisms that are known to account for TKI resistance in primary CML-LSCs and to focus on the potential solutions that can circumvent these resistances, in particular those that have been, or will be tested in clinical trials.

scholarly journals Shedding Light on Targeting Chronic Myeloid Leukemia Stem Cells

2021 ◽  
Vol 10 (24) ◽  
pp. 5805
Author(s):  
Mohammad Houshmand ◽  
Alireza Kazemi ◽  
Ali Anjam Najmedini ◽  
Muhammad Shahzad Ali ◽  
Valentina Gaidano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Term Treatment ◽  
Leukemia Stem Cells ◽  
Hematopoietic Stem ◽  
Long Term Treatment ◽  
Treatment Free Remission

Chronic myeloid leukemia stem cells (CML LSCs) are a rare and quiescent population that are resistant to tyrosine kinase inhibitors (TKI). When TKI therapy is discontinued in CML patients in deep, sustained and apparently stable molecular remission, these cells in approximately half of the cases restart to grow, resuming the leukemic process. The elimination of these TKI resistant leukemic stem cells is therefore an essential step in increasing the percentage of those patients who can reach a successful long-term treatment free remission (TFR). The understanding of the biology of the LSCs and the identification of the differences, phenotypic and/or metabolic, that could eventually allow them to be distinguished from the normal hematopoietic stem cells (HSCs) are therefore important steps in designing strategies to target LSCs in a rather selective way, sparing the normal counterparts.

scholarly journals Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

Blood ◽  
2012 ◽  
Vol 119 (6) ◽  
pp. 1501-1510 ◽  
Author(s):  
Ashley Hamilton ◽  
G. Vignir Helgason ◽  
Mirle Schemionek ◽  
Bin Zhang ◽  
Svetlana Myssina ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Inhibition Of Proliferation ◽  
Abl Kinase

Abstract Recent evidence suggests chronic myeloid leukemia (CML) stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiologic growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%; however, the surviving fraction was enriched for primitive leukemic cells capable of growth in a long-term culture-initiating cell assay and expansion on removal of dasatinib and addition of growth factors. Together, these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance.

Long-term treatment-free remission of chronic myeloid leukemia with falling levels of residual leukemic cells

Leukemia ◽  
2018 ◽  
Vol 32 (12) ◽  
pp. 2572-2579 ◽  
Author(s):  
David M. Ross ◽  
◽  
Ilaria S. Pagani ◽  
Naranie Shanmuganathan ◽  
Chung H. Kok ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Term Treatment ◽  
Leukemic Cells ◽  
Long Term Treatment ◽  
Treatment Free Remission

Rapid Reduction of Chronic Myeloid Leukemia Stem Cells After Treatment with Second-Generation BCR-ABL Kinase Inhibitors, Dasatinib and Nilotinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4457-4457
Author(s):  
Yosuke Minami ◽  
Akihiro Abe ◽  
Yuka Nomura ◽  
Miho Minami ◽  
Yachiyo Kuwatsuka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Second Generation ◽  
Kinase Inhibitors ◽  
Newly Diagnosed ◽  
Rapid Reduction ◽  
Population Total ◽  
Significant Difference

Abstract Abstract 4457 Chronic myeloid leukemia (CML) is effectively treated with imatinib (IM), however, several mathematical models and ex vivo-examinations suggested that IM-therapy does not eradicate BCR-ABL-positive hematopoietic stem cells (HSC). We prospectively (0, 3, 6 and 12 months after IM-therapy) investigated 16 newly diagnosed and 22 long-term followed CML-chronic phase (CP) cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) (Figure 1) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1+, HSC/Thy-1–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM-therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1+ and HSC/Thy-1–) tended to be more retentive than other populations while gradual reduction was observed during the first 12 months in all populations. And discrepancy of minimum residual diseases (MRD) between the HSC populations and other populations was larger in patients after longer IM-therapy. In evaluating properties of CML stem cells and other markers, we observed irrelevant distribution of side population (SP) and expressions of ABC transporters (ABCB1 and ABCG2) in comparison with CD34/38 expression. We also prospectively investigated BCR-ABL transcripts in each population of 23 IM-resistant or -intolerant CML-CP cases and one newly diagnosed CML-accelerated phase (AP) case during treatment with second-generation tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34+CD38–) during the first 6 months and there was no significant difference of MRD among each population in optimal responders to 2nd TKIs-therapy. In the stromal co-culturing system using primary cells and leukemic NOD/SCID/IL2rgnull (NOG) mice xenotransplanted with Ph+ leukemia cells, retention of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ population after IM-treatment were observed and cell death mechanisms after treatment with 2nd TKIs are also under investigation. These results imply that therapy with 2nd TKIs could be a promising approach for quick and efficient reduction of the CML stem cells and cure of disease. Figure 1 Figure 1. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Induced Pluripotent Stem Cells (iPSC) From Chronic Myeloid Leukemia: Study of BCR-ABL Addiction and Effect of Tyrosine Kinase Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3754-3754 ◽  
Author(s):  
Aurélie Bedel ◽  
Francois Moreau-Gaudry ◽  
Jean- Max Pasquet ◽  
Miguel Taillepierre ◽  
Éric Lippert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Leukemic Stem Cells ◽  
Rt Pcr ◽  
Reprogramming Factors

Abstract Abstract 3754 The tyrosine kinase inhibitors (TKI) such as imatinib, by suppressing BCR-ABL oncogene activity, are an effective therapy for chronic myeloid leukemia disease (CML). However, the majority of patients achieving remission with TKI continue have molecular evidence of persistent disease. In addition, we have reported that for patients who achieved a sustained complete molecular remission, 60% of them relapse after discontinuation of imatinib. Various mechanisms have been proposed to explain disease persistence and disease recurrence. One of the hypotheses is that primitive leukemic stem cells can survive in the presence of TKI. Little is known about the stem cells survival due to technical difficulties (small and poorly defined primary populations). Understanding the mechanisms by which these cells survive to TKI therapy will be critical to devising strategy aimed to their elimination. We propose to generate iPSC derived from CD34+ blood cells isolated from CML patient (CML-iPSC), as a model for study leukemic stem cells survival in the presence of TKI and study the mechanism of TKI resistance of the stem cells. Primary CD34+ CML patient cells were transduced by 2 excisable lentiviral vectors (both flanked by two LoxP sites), one expressing three reprogramming factors (OCT4-SOX2-KLF4) and another one with c-MYC and a shRNA against TP53. Twenty-one days after co-transduction, CML-iPSC colonies were picked and five iPS clones were characterized (expression of pluripotency markers by RT-PCR (DPPA4, NANOG, CRIPTO) and immunofluorescence (NANOG, SSEA-4, TRA1-60)). Efficiency of reprogrammation was low compared to cord blood CD34+ control cells (0.01% vs 0.1%, respectively), and delayed (21 days vs 14 days). Philadelphia chromosome (Ph) positive was observed in 4/5 clones after cytogenetic analysis. Expression of BCR-ABL (Western-blot and RT-PCR) was present at various levels. Interestingly, 1/5 clone was generated from non-leukemic cell (Ph negative) and was used as internal control for the following function assays. We used these 5 CML-iPSC clones to study their behavior in presence of TKI. All CML-iPSC clones survived to escalating concentration of imatinib (0 to 20μM) and ponatinib (0 to 50nM) for 6 days. To understand if the CML-iPSC survival was due to resistance or independence mechanisms, we performed western blot analysis of TKI targets. BCR-ABL activity was inhibited under TKI exposure (dephosphorylations of BCR/ABL and of Crkl). In order to check whether survival was due to the expression of reprogramming factors, we excised the gene cassettes by an Adenovirus expressing CRE recombinase. After proviral excision and subcloning, excised CML-iPSC continued to survive to TKI exposure. Taken together, these results demonstrate that CML-iPSC survival do not depend on BCR-ABL (oncogene independence). Upon induction of hematopoietic differentiation, CML-iPSC were able to efficiently generate progenitors of hematopoietic lineages (up to 40% of CD45+) and colony forming units in methylcellulose. TKI effect on iPSC-derived hematopoietic progenitors, to analyze the putative recovery of TKI sensibility compared to primitive CML blood cells from the same patient, are in progress. We conclude that reprogrammation of CD34BCR-ABL+ cells from CML patient is possible and that CML-iPSC lost the BCR-ABL dependency and became resistant to TKI. A specific differentiated epigenetic cell state is probably needed to maintain BCR-ABL dependency. CML-iPSC can be used to study mechanisms by which leukemic stem cells survive to TKI therapy and is a promising tool for testing and screening new therapeutic target reducing leukemic stem cell survival. Disclosures: Mahon: Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizzer: Honoraria.

Molecular Characterization of the Leukemic Niche in Chronic Myeloid Leukemia (CML) and Evaluation of a Leukemia / Niche Cross-Talk

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 908-908
Author(s):  
Djamel Aggoune ◽  
Nathalie Sorel ◽  
Sanaa El Marsafy ◽  
Marie Laure Bonnet ◽  
Denis Clay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mrna Expression ◽  
Expression Pattern ◽  
Myeloid Leukemia ◽  
Gene Expression Pattern ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Whole Genome Sequencing Of Chronic Myeloid Leukemia (CML)-Derived Induced Pluripotent Stem Cells (iPSC) Reveals Faithful Genocopying Of Highly Mutated Primary Leukemic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 514-514
Author(s):  
Ivan Sloma ◽  
Maria Teresa Mitjavila-Garcia ◽  
Olivier Feraud ◽  
Noufissa Oudrhiri ◽  
Lucie Tosca ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Pluripotent Stem Cells ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Jak2 V617f ◽  
Leukemic Cells ◽  
Whole Genome ◽  
Induced Pluripotent ◽  
Short Indels

Abstract Genetic instability is a hallmark of chronic myeloid leukemia (CML). Recently, several major abnormalities in DNA repair mechanisms have been identified in primitive CML cells that likely explain the additional mutations these cells develop leading to their selective growth under tyrosine kinase inhibitor (TKI) therapies. It seems likely that such mechanisms also underlie disease progression in CML. However, an understanding of the specific somatic mutations involved and investigations of their resulting effects on the biological behavior of primary sources of primitive chronic phase (CP) CML cells is extremely challenging. As an alternative approach, we have now explored the possibility of applying whole genome sequencing (WGS) to induced pluripotent stem cells (iPSCs) derived from primitive CML cells to determine if such iPSCs, genocopy the mutations present in the diagnostic sample from which they were generated and whether primitive hematopoietic cells derived from these iPSCs might be useful for future drug screening experiments. To this end, we chosen a CML patient whose CP clonogenic cells contained both the Ph1 chromosome and the JAK2 V617F mutation and whose disease progressed into an accelerated phase (AP) during TKI therapy. iPSC were generated from leukemic cells obtained at the time of AP using Oct4, Sox2, Klf4 and c-Myc gene transfer. The presence of both BCR-ABL and JAK2 V617F was confirmed in 24/24 iPSC colonies. A control iPSC line negative for both genes was similarly established from the patient’s CD34+CD31+ endothelial progenitors purified from peripheral blood. We then performed WGS on DNA prepared from the leukemic cells obtained at diagnosis of CP (CML 006), the AP cell-derived iPSCs (PB34), and the control non-leukemic iPSCs (PB13), using a HighSeq Illumina platform. WGS revealed 845,175 somatic SNVs and 68,817 somatic short Indels in the CP leukemic cells at diagnosis that were not present in the non-leukemic iPSCs (PB13). 49,225 of these SNVs and 11,665 of the short Indels were novel (absent in the dbSNP database), and 419 were found in the COSMIC database. We identified 274 novel SNVs (3 missense, 161 nonsense, 108 synonymous and 2 splice site mutations) and 46 short Indels (19 insertions and 27 deletions). Most of the novel coding SNVs and Indels were heterozygous and an estimation of the variant allele frequency indicated these were present in virtually all leukemic cells. In addition to the JAK2 V617F mutation that was present at diagnosis, we found a novel frame shift mutation in exon 12 of ASXL1 gene (p.S871YfsX5) leading to protein truncation, a genetic event that has also been associated with myeloproliferative neoplasms (MPNs) and AML. We also identified several novel SNVs predicted by SIFT, Provean and PolyPhen-2 algorithms to be deleterious for protein structure. These novel mutations were found in genes relevant for the pathophysiology of MPNs, including the catenin (CTNNA1 R204C, and AIDA K235T), RAS (RREB1 P789T), autophagy (ULK1 R553C) cellular antioxidant defense (GSR S293C), RNA nuclear transport (NUP160 start loss) pathways. Individual sequencing confirmed the presence of these mutations in PB34 and their absence in PB13 (non-leukemic iPSC). We next compared the sequence data from the AP leukemic cell-derived iPSCs (PB34) with the diagnostic data (CML006). This analysis showed only 799 additional somatic SNVs and 96 new short Indels compared with those already evident in the cells present at diagnosis. Only 4 (3 non synonymous and 1 synonymous) SNVs and no Indels were found in exons. These mutations could have appeared during the application of the reprogramming process to the AP leukemic cell-derived iPSCs; none was an obvious contributor to MPN pathophysiology. Finally, we showed that day16 embryoid bodies derived from the PB34 iPSCs contained expected numbers of CD34+ cells (18±11%, n=6) and BCR-ABL-expressing hematopoietic colony-forming cells (CFCs, 143±64 / 105 cells, n= 6). These CFCs showed a slight inhibitory response to imatinib (54±15% colonies obtained in 1 µM IM, n=4) whereas a combination of IM and Pimozide (a STAT5 phosphorylation inhibitor), reduced survival another ∼10-fold. In conclusion, we have provided proof-of-principle results illustrating the potential of iPSC technology in combination with WGS to dissect the clonal evolution of disease progression in CML and develop patient specific drug screens that could build on this data. Disclosures: Turhan: BMS, Novartis: Honoraria, Research Funding.

Targeting of heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) in leukemic cells in chronic myeloid leukemia: a novel approach to overcome resistance against imatinib

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2200-2210 ◽  
Author(s):  
Matthias Mayerhofer ◽  
Karoline V. Gleixner ◽  
Julia Mayerhofer ◽  
Gregor Hoermann ◽  
Eva Jaeger ◽  
...  
Keyword(s):  
Heat Shock ◽  
Chronic Myeloid Leukemia ◽  
Heat Shock Protein ◽  
Heme Oxygenase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Leukemic Cells ◽  
Heme Oxygenase 1 ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle–encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1–targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.

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