Survival In Chronic Myelogenous Leukemia Patients Research Articles

The Discovery of Novel BCR-ABL Tyrosine Kinase Inhibitors Using a Pharmacophore Modeling and Virtual Screening Approach.

Chronic myelogenous leukemia (CML) typically results from a reciprocal translocation between chromosomes 9 and 22 to produce the bcr-abl oncogene that when translated, yields the p210 BCR-ABL protein in more than 90% of all CML patients. This protein has constitutive tyrosine kinase activity that activates numerous downstream pathways that ultimately produces uncontrolled myeloid proliferation. Although the use of the BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have increased the overall survival of CML patients, their use is limited by drug resistance and severe adverse effects. Therefore, there is the need to develop novel compounds that can overcome these problems that limit the use of these drugs. Therefore, in this study, we sought to find novel compounds using Hypogen and Hiphip pharmacophore models based on the structures of clinically approved BCR-ABL TKIs. We also used optimal pharmacophore models such as three-dimensional queries to screen the ZINC database to search for potential BCR-ABL inhibitors. The hit compounds were further screened using Lipinski’s rule of five, ADMET and molecular docking, and the efficacy of the hit compounds was evaluated. Our in vitro results indicated that compound ZINC21710815 significantly inhibited the proliferation of K562, BaF3/WT, and BaF3/T315I leukemia cells by inducing cell cycle arrest. The compound ZINC21710815 decreased the expression of p-BCR-ABL, STAT5, and Crkl and produced apoptosis and autophagy. Our results suggest that ZINC21710815 may be a potential BCR-ABL inhibitor that should undergo in vivo evaluation.

Correlation of Expression of CD25 in Hematopoietic Stem/Progenitor Cell Fraction of Bone Marrow Cells with Response to Tyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia Patients

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs continues to improve the depth of response and overall survival of CML patients, but the life-long use of TKI is known to be associated with late complications such as cardiovascular events as well as heavy financial burden, and thus impairs the quality of life. To overcome these issues, many studies evaluating the possibility of TKI discontinuation have been ongoing worldwide. In order to achieve durable treatment-free remission, it is crucial to understand the dynamics of CML-leukemia initiating cells (LICs). We previously reported that CD25 was highly expressed in murine and human CML-LICs (Kobayashi CI et al., Blood, 2014). The aim of this study was to assess whether the proportion of CD25 positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to stop TKI therapy.Methods: Bone marrow samples were obtained from patients with CML in chronic phase who were diagnosed and have been treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25 positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction was evaluated by flow cytometry using FITC-labeled anti-CD34, PE- labeled anti-CD38 and APC-labeled anti-CD25 antibodies. The response to TKIs at the time of evaluation was determined according to the previous report (Yoshida C et al., Int J Clin Oncol, 2012): complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL by quantitative and qualitative PCR.Results: Bone marrow samples obtained from 95 patients were evaluated (median age 53 years old; male/female, 67/28). Analysis was performed prior to TKI exposure in nine patients and under TKI therapy including 2nd generation TKI in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 22 patients were treatment free because they enrolled in a clinical trial of TKI discontinuation. The proportion of CD25 positive cells in HSPC fraction significantly decreased in patients with prior TKI exposure relative to patients at diagnosis (n=86; Mean 4.2%, SD 7.0% vs n=9; Mean 22.4%, SD 11.3%, P<0.01). In addition, the proportion of CD25 positive cells in HSPC fraction significantly correlated the level of quantitative PCR (Figure, P<0.0001), and MMR was also dividable from CMR by the proportion of CD25 positive cells (MMR; n=29, Mean 4.6%, SD 4.8% vs CMR; n=49, Mean 2.6%, SD 2.5%, P<0.05). The expression of CD25 was still detected in the majority of patients who achieved CMR, including those who sustained CMR after the discontinuation of TKIs.Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the response to TKI therapy, and may serve as an asset to select patients who are likely to achieve durable treatment-free survival. [Display omitted] DisclosuresKarigane:Celgene: Honoraria. Sakurai:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Mitsuhashi:LSI Medience: Consultancy. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Knockdown (KD) of Mir-126 Expression Enhances Tyrosine Kinase Inhibitor (TKI)-Mediated Targeting of Chronic Myelogenous Leukemia (CML) Stem Cells

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. The long-term goal of our research is to improve the understanding of molecular mechanisms regulating LSC growth and develop effective mechanism-based therapeutic strategies to target LSC in CML. MicroRNAs (miRNAs) are short non-coding RNAs that regulate translation of target protein-coding messenger RNAs (mRNAs) and levels of the corresponding encoded proteins. Among distinct miRNAs, miR-126-3p (miR-126) is expressed in normal hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) and plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo (Cell Stem Cell, 2012). We previously reported that miR-126 expression varies among AML patients; higher level of miR-126 associated with poor outcome, a LSC-associated gene expression signature and increased LSC quiescence; and miR-126 down-regulation decreased LSC self-renewal activity in serial transplant experiments (Leukemia, 2015). Here, we report that the more primitive LSK (Lin-Sca-1+Kit+) from SCL-tTA-BCR/ABL mouse model of CML had higher miR-126 expression compared to more differentiated subpopulations, i.e. common myeloid progenitors (CMP) (3.2 fold) and granulocyte-macrophage progenitors (GMP) (6.4 fold). Among LSK subpopulations, LSC (Flt3-CD150+CD48- LSK) demonstrated the highest miR-126 levels (P<0.01). Furthermore, quiescent (Hoechst-Pyronin-) LSC demonstrated higher miR-126 expression (2.6 fold, p<0.01), generated significantly higher long-term engraftment of donor CML cells in recipient mice (58.6±4.4% vs 33±3.4% in BM at 16 wks after transplanted with 100 LSC/mouse, p=0.003) and enhanced leukemogenic capacity (5 out of 9 mice receiving quiescent LSC developed CML within 14 wks after transplantation vs. none of 10 mice receiving proliferating LSC) compared with proliferating (Hoechst+/-Pyronin+) LSC. Human primary untreated chronic phase (CP) CML CD34+CD38- primitive progenitors also showed higher miR-126 expression compared with CD34+CD38+ committed progenitors. KD of miR-126 in LSK cells of CML mouse model and in primary CML CD34+CD38- cells by using either miRZip-126-3p anti-miR lentivirus (System Biosciences) or a novel, myeloid cell-specific CpG-miR-126 oligonucleotide (ODN) inhibitor increased LSC cell-cycle entry as demonstrated by increased Ki67 staining (p<0.05) and EdU labeling (p<0.05), but affected apoptosis only slightly compared to scramble-treated controls. More importantly, miR-126 KD either by lentivirus or CpG-miR-126 ODN inhibitor combined with Nilotinib (NIL) significantly increased apoptosis of LSC compared with NIL alone (34% vs 19%; p=0.01 for lentivirus KD; and 40% vs 35%; p=0.03 for CpG-miR-126 inhibitor KD), resulting in significant reduction of cell growth and colony forming cell (CFC) frequency in methylcellulose progenitor assays. Conversely, enforced miR-126 expression by miR-126 precursor lentivirus (System Biosciences) impaired cell-cycle entry (28% vs 33%; p=0.05) and led to significantly reduced apoptosis (27% vs 19%; P=0.01) in NIL (5µM) treated CML CD34+CD38- cells compared to cells treated with control lentivirus and NIL. We further investigated the effect of miR-126 KD on growth of primary CML LSC and therapeutic response in vivo. CP CML CD34+ cells were cultured with CpG-scrambled RNA or CpG-miR-126 ODN inhibitor (500nM), in the presence or absence of NIL (5µM) for 4 days, and then transplanted into irradiated NSG mice. While the experiment is still ongoing, we have already observed a significantly reduced engraftment in blood at 4 wks in recipient mice receiving cells treated with CpG-miR-126 inhibitor and NIL compared with those receiving cells treated with CpG-scrambled RNA and NIL (0.16±0.03% vs 0.4±0.04%; P=0.01). Final results of this in vivo experiment and in vivo treatment of SCLtTA/BCR-ABL mice with CpG-miR-126 inhibitor and NIL will be reported at the meeting. Altogether, these observations suggest that miR-126 play a role in CML LSC homeostasis and down-regulation of miR-126 may decrease CML LSC quiescence, increase LSC proliferation and in turn enhance their sensitivity to TKI. miR-126 therefore may represent a novel therapeutic target in CML. DisclosuresStein:Amgen: Speakers Bureau. Snyder:BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang: Research Funding; Amgen: Consultancy.

Rac GTPases as key regulators of p210-BCR-ABL-dependent leukemogenesis

Chronic myelogenous leukemia (CML) is a malignant disease characterized by expression of p210-BCR-ABL, the product of the Philadelphia chromosome. Survival of CML patients has been significantly improved with the introduction of tyrosine kinase inhibitors that induce long-term hematologic remissions. However, mounting evidence indicates that the use of a single tyrosine kinase inhibitor does not cure this disease due to the persistence of p210-BCR-ABL at the molecular level or the acquired resistance in the stem cell compartment to individual inhibitors. We have recently shown in a murine model that deficiency of the Rho GTPases Rac1 and Rac2 significantly reduces p210-BCR-ABL-mediated proliferation in vitro and myeloproliferative disease in vivo, suggesting Rac as a potential therapeutic target in p210-BCR-ABL-induced disease. This target has been further validated using a first-generation Rac-specific small molecule inhibitor. In this review we describe the role of Rac GTPases in p210-BCR-ABL-induced leukemogenesis and explore the possibility of combinatorial therapies that include tyrosine kinase inhibitor(s) and Rac GTPase inhibitors in the treatment of CML.

Impact of Complete Kariotypic Remission (CKR) and Sokal Risk on Overall Survival in Chronic Myelogenous Leukemia (CML) Patients: A Monocentric Experience.

Introduction Progressive improvement has been observed in CKR and survival of CML patients (pts) in response to interferon (IFN)a-based regimens, or imatinib. The purpose of this study is the evaluation of:rate of, time to and duration of CKR in accordance to first line therapy employed and Sokal score;impact on overall survival of CKR, and Sokal score, separately considered or combined together.Patients. 109Ph'+ and 5Ph'−, (BCR-ABL positive), CML pts were treated at diagnosis with allogeneic transplantation (3 pts), hydroxyurea (HU) (19 pts), INFa (51pts, G1), INFa associated with ARA-C (20 pts, G2), imatinib alone (18 pts, G3), or imatinib combined with INFa (3 pts, G4). INFa was employed as second line therapy in 12 pts initially treated with hydroxyurea (G5), while INFa/ARA-C combination or imatinib alone was given to 24 (G6) and 23 (G7) pts with de novo or acquired resistance or intolerance to INFa. Third line therapy, consisting of the combination of imatinib with IFNa, was employed in 11 (G8) pts with no CKR (5 pts) or in complete cytogenetic, but not in molecular remission (6 pts).Results. 40 of 94 Ph'+ evaluable non-allotransplanted pts obtained one or more (overall 47) CKRs to INFa-based regimens or imatinib. CKR rate, median time to CKR and response duration are shown in table 1. In the analysis according to Sokal score 82/94 pts, with complete prognostic data at diagnosis, were included. The percentage of responders was higher in the low compared to the non-low Sokal risk group (57% vs. 31%). Irrespective of the treatment, median duration value of the first CKR was also better in the former [18+mths(1–64)] than in the latter group [6mths(2–54)] with 16 vs.4 pts still in first or subsequent remission. Overall survival for CKRs was 68+mths(5–275) vs. 52mths(5–270) for CKRs with 35 vs. 6 pts still alive respectively. Overall survival according to Sokal score at diagnosis was 61+ mths(5–275) for low vs 53mths(5–212) for non-low risk patients. The impact on survival of CKR and Sokal risk were then analyzed simultaneously. The median survival of 27 CKRs and 20 not CKRs with low Sokal risk were 61+mths(5–275)and 63 mths(14–270) respectively as compared to 73+mths(11–212) of 11 CKRs and 36mths(5–139) of 24 not CKRs with unfavourable characteristics at diagnosis. The number of patients still alive in these 4 groups were 24/27, 3/20, 8/11, 3/24 respectively at the time when this analysis was performed.Conclusions. The present data not only confirm the effectiveness of imatinib-over the INFa-based regimens in inducing CKR, but also suggest that response to treatment may be better than Sokal risk in predicting patient survival.Rate,time to and duration of CKR according to treatmentPT GroupCKR rate(%)time to CKR( months)Duration of CKR (months)N° of pts in cCKRMedianRangeMedianRangeG11716(3–28)6(2–53)0G23011(3–24)15,5(2–33)0G3856(2–14)10+(1–44)11G41004(4–5)48(2–50)1G500G699,5(7–12)40,5(17–64)1G7549(4–38)21,5+(3–54)6G8803(2–4)26+(4–48)9

Complete cytogenetic and molecular responses to interferon-alpha-based therapy for chronic myelogenous leukemia are associated with excellent long-term prognosis.

Little is known regarding long-term prognosis among patients with Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) who achieve a complete cytogenetic response (0% Ph-positive cells) after treatment with interferon-alpha. The authors analyzed 512 patients with Ph-positive, early chronic-phase CML who were treated with interferon-based therapies between 1981-1995 for the incidence and durability of complete cytogenetic response, and in relation to long-term prognosis. One hundred forty patients (27%) achieved a complete cytogenetic response. Their 10-year survival rate was 78%. At the time of last follow-up, 44 patients (31%, 9% of the total) were alive, 21 in first and 23 in second durable complete cytogenetic response (median, 127 months; range, 88-191 months); 39 patients had not received any therapy for a median of 50 months (range, 11-139 months). Analysis by reverse transcriptase-polymerase chain reaction in 78 patients during complete cytogenetic response showed 46 who had achieved at least 1 complete molecular response. Five of these 78 patients had died by the time of last follow-up, but only 2 had died of disease-specific causes. Recurrence rates were significantly lower and cytogenetic response durations were significantly longer among patients who achieved at least one complete molecular response. Achieving a complete cytogenetic or molecular response after therapy with interferon-alpha appears to be associated with excellent long-term prognosis. Approximately 10% of patients reportedly can achieve durable complete cytogenetic response, with or without continuation of interferon. This finding emphasizes the potential of long-term event-free survival in CML patients outside the context of allogeneic stem cell transplantation, which may be improved with new therapies such as imatinib mesylate.