Deregulated expression of miR-29a-3p, miR-494-3p and miR-660-5p affects sensitivity to tyrosine kinase inhibitors in CML leukemic stem cells

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm generated by reciprocal chromosomal translocation, t (9; 22) (q34; q11) in the transformed hematopoietic stem cell. Tyrosine kinase inhibitors (TKIs) target the mature proliferating BCR-ABL cells, the major CML driver, and increase overall and disease-free survival. However, mutant clones, pre-existing or due to therapy, develop resistance against TKIs. BCR-ABL1 oncoprotein activates various molecular pathways including the RAS/RAF/MEK/ERK pathway, JAK2/STAT pathway, and PI3K/AKT/mTOR pathway. Stimulation of these pathways in TKI resistant CML patients, make them a new target. Moreover, a small proportion of CML cells, leukemic stem cells (LSCs), persist during the TKI therapy and sustain the disease in the patient. Engraftment of LSCs in the bone marrow niche and dysregulation of miRNA participate greatly in the TKI resistance. Current efforts are needed for determining the reason behind TKI resistance, identification, and elimination of CML LSC might be of great need for cancer cure.

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An Extended Mathematical Model of Pathophysiology and Response to Treatment in Chronic Myelogenous Leukemia

Blood ◽

10.1182/blood.v112.11.4220.4220 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4220-4220

Author(s):

Yasuhito Nannya ◽

Yoichi Imai ◽

Akira Hangaishi ◽

Mineo Kurokawa

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Chronic Myelogenous Leukemia ◽

Peripheral Blood ◽

Kinase Inhibitors ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells

Abstract Chronic myelogenous leukemia (CML) is a malignant clonal disorder of hematopoietic stem cells that results in increase in myeloid, erythroid cells, and platelets in the peripheral blood and marked myeloid hyperplasia in the bone marrow. This disorder is characterized by the specific cytogenetic abnormality, the Philadelphia (Ph) chromosome, which results from a balanced translocation between the long arms of chromosomes 9 and 22, generating the bcr/abl chimeric gene that expresses an abnormal fusion protein with altered tyrosine kinase activity. Imatinib mesylate (IM, Gleevec, Novartis, Basel, Switzerland), is a potent and selective competitive inhibitor of the BCR-ABL protein tyrosine kinase and has shown to induce a high rate of cytogenetic and hematologic response in patients with chronic phase (CP) CML both as initial therapy and as secondary therapy after previous interferon therapy failed. Because the pathophysiology of CML and the mechanism for the clinical effects by IM is relatively uniform among patients, simplification and generalization with mathematical models have been proposed and they have excellently simulated the regression of leukemic cells by IM therapy and the regrowth of CML cells after appearance of IM-resistant clones. These models are based on the assumption that the transition rate of leukemic stem cells or precursor cells to more differentiated fractions are profoundly diminished by the administration of IM. This assumption is sufficient to explain the response as long as the observation period is short. In contrast, the issue regarding the influence of IM on the self-reproduction rate of leukemic stem cells was not focused on in these models because this issue had little effect on short-term outcomes with IM. After a decade since the appearance of IM, accumulated observations of CML patients treated with IM revealed long-term effectiveness; novel transformations to accelerate phase or blastic crisis are rarely observed in patients who continue to receive 400mg/day of IM for five or six years. Our aim is to clarify the effect of IM on leukemic stem cell fractions by extending and modifying the existing models so that they are compatible with actual long-term outcomes of IM therapy. First, we demonstrated that sustained effectiveness of IM for over six years cannot be achieved unless a stem cell fraction of CML is decremented by IM. In order to estimate the degree of stem cell attack by IM, we computed the rate of novel generation of IM-resistant clones before and after IM administration. In this model, we presumed that this rate is proportional to the accumulated number of self-duplication of leukemic stem cells. In order to simulate the actual observation that the clonal evolution decrease annually after IM administration, we illustrated that the rate of self duplication is depleted to at one fourth or less with IM compared to without IM. With this simulation, we show that the tyrosine kinase inhibitors can eradicate malignant cells thus leading to the radical cure of the disease. We also showed that the achievement of major molecular response (MMR; defined as at least three-log reduction of bcr/abl positive clones in the peripheral blood) at the 18th month of IM therapy is roughly associated with the absence of resistant clones at the moment of IM administration, and is obviously linked to successful therapy of CML after IM therapy is launched. This provides the supportive evidence of the previously reported observation that MMR at 18th month is associated with long-term effectiveness. Our model underscores the significance of prompt elimination of leukemic stem cells in order to diminish the generation of novel resistant clones and accomplish complete cure of CML. Development of the evaluation system to quantify residual leukemic stem cells would verify this hypothesis and pursuit to maximal response including early administration of second-generation tyrosine kinase inhibitors would be justified.

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Discontinuation of tyrosine kinase inhibitors and new approaches to target leukemic stem cells: Treatment-free remission as a new goal in chronic myeloid leukemia

Cancer Letters ◽

10.1016/j.canlet.2014.01.033 ◽

2014 ◽

Vol 347 (1) ◽

pp. 22-28 ◽

Cited By ~ 13

Author(s):

Massimo Breccia ◽

Giuliana Alimena

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Leukemic Stem Cells ◽

New Approaches ◽

Treatment Free Remission

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Resistance to tyrosine kinase inhibitors and novel insights into genomic instability of chronic myeloid leukemia stem cells

Chronic Myeloid Leukemia ◽

10.3109/9781842145784.009 ◽

2013 ◽

pp. 108-117

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Genomic Instability ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Leukemia Stem Cells

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Inhibition of heat shock protein 90 prolongs survival of mice with BCR-ABL-T315I–induced leukemia and suppresses leukemic stem cells

Blood ◽

10.1182/blood-2006-10-054098 ◽

2007 ◽

Vol 110 (2) ◽

pp. 678-685 ◽

Cited By ~ 108

Author(s):

Cong Peng ◽

Julia Brain ◽

Yiguo Hu ◽

Ami Goodrich ◽

Linghong Kong ◽

...

Keyword(s):

Stem Cells ◽

Heat Shock ◽

Heat Shock Protein ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Heat Shock Protein 90 ◽

Hsp90 Inhibitor ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells

Abstract Development of kinase domain mutations is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. A particularly challenging example is found in Philadelphia chromosome–positive chronic myelogenous leukemia (CML) where all available kinase inhibitors in clinic are ineffective against the BCR-ABL mutant, T315I. As an alternative approach to kinase inhibition, an orally administered heat shock protein 90 (Hsp90) inhibitor, IPI-504, was evaluated in a murine model of CML. Treatment with IPI-504 resulted in BCR-ABL protein degradation, decreased numbers of leukemia stem cells, and prolonged survival of leukemic mice bearing the T315I mutation. Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the wild-type (WT) clones in mice. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice simultaneously bearing both WT and T315I leukemic cells. These results provide a rationale for use of an Hsp90 inhibitor as a first-line treatment in CML by inhibiting leukemia stem cells and preventing the emergence of imatinib-resistant clones in patients. Rather than inhibiting kinase activity, elimination of mutant kinases provides a new therapeutic strategy for treating BCR-ABL–induced leukemia as well as other cancers resistant to treatment with tyrosine kinase inhibitors.

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Role of New Tyrosine Kinase Inhibitors in Osteoblastogenesis

Blood ◽

10.1182/blood.v112.11.4751.4751 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4751-4751

Author(s):

Daniele Tibullo ◽

Cesarina Giallongo ◽

Piera La Cava ◽

Provvidenza Guagliardo ◽

Maide Cavalli ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Tyrosine Kinase ◽

Osteogenic Differentiation ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Osteogenic Medium ◽

Standard Medium ◽

Alizarin Red

Abstract It has been reported that imatinib mesylate (IM) may affect bone tissue remodeling mainly by both an inhibitory activity on osteoclastogenesis and an induction of osteoblastogenesis. Dasatinib (DA) and Nilotinib (NI) are new generation tyrosine kinase inhibitors presently approved for chronic myeloid leukemia patients after imatinib failure. We therefore evaluated possible effects of DA and NI on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BM-MSCs). BM-MSCs are multipotent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors, isolated by density gradient (mononuclear fraction) and cultured either in standard medium (SM) or in osteogenic medium (OM) (0.2 mM ascorbic acid, 0.1 μm dexamethasone and 10 mM β-glycerophosphate) with or without DA 2nM or NI 100nM. Osteogenic differentiation of hBM-MSCs was evaluated by changes in morphology, presence of mineralized nodules (evidenced by Alizarin red) and expression of osteoblast-associated genes such as osteocalcin (OCN), RUNX2 and Bone morphogenetic protein (BMP-2) evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by Scion Image. After 21days of culture, in comparison to control cultures, hBM-MSCs placed in OM, DA, NI and DA+OM, NI+OM exhibited changes in cell morphology from a spindle-shaped fibroblastic appearance to a rounder more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization). Table I indicates mRNA expression of osteogenic markers in different culture conditions and shows that both DA and NI alone or in combination with OM, increase RUNX2, OCN, and BMP-2 expression. SM DA NI OM DA + OM NI + OM SM= standard medium, OM= osteogenic medium, DA= dasatinib, NI= nilotinib In summary, our data show that both DA and NI, as already reported IM, may induce osteogenic differentiation of mesenchymal cells thus indicating that they potentially favour osteoblastogenesis. RUNX2 1,59 0,20 2,09 0,16 4,2 0,31 2,86 0,25 4,41 0,41 4,18 0,24 OCN 2,57 0,28 3,2 0,14 3,14 0,09 3,59 0,17 3,6 0,28 3,62 0,25 BMP-2 1,55 0,19 2,27 0,17 4,16 0,27 2,84 0,28 4,43 0,30 4,21 0,30

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

Blood ◽

10.1182/blood.v118.21.3754.3754 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3754-3754 ◽

Cited By ~ 1

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.

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