Pathophysiology Of Chronic Myeloid Leukemia Research Articles

Estimation of miRNA-183 expression and TGF-β level in Chronic Myeloid Leukemia

Background: MicroRNA-183 and Transforming Growth Factor Beta (TGF-β) have emerged as significant players in the pathophysiology of Chronic Myeloid Leukemia (CML). Research indicates that miRNA-183 may play a role in the regulation of cell proliferation and apoptosis in CML cells, potentially influencing disease progression and response to therapy. Elevated levels of miRNA-183 have been associated with poor prognosis, suggesting it could serve as a biomarker for disease severity. Objective: In this study, we aimed to analyze the expression of miR-183 and TGF- β level and investigate the relationship between miR-183 and level of TGF- β in CML patients. Material and Methods: The study has been carried out on 60 Iraqi patients (37 males and 23 females at age range 17 - 69 year) with chronic myeloid leukemia at chronic phase, who are referred to the National Center of Hematology/ Mustansiriyah University, Baghdad during the period from January to November 2023. Twenty of them are newly diagnosed (T0), and the other 40 patients are under treatment with TKIs (T+), Thirty age and gender-matched healthy subjects were enrolled to act as control group. TGF-β level estimates by ELISA while micrRNA-183 detection using RT- PCR. Conclusion: The elevated levels of TGF-β and miRNA-183 in CML patients, coupled with their positive correlation.

JAK2 as Predictor of Therapeutic Response in Patients with Chronic Myeloid Leukemia Treated with Imatinib.

Chronic myeloid leukemia (CML) or chronic granulocytic leukemia is a myeloproliferative neoplasm indicated by the presence of the Philadelphia (Ph+) chromosome. First-line tyrosine kinase inhibitor, imatinib, is the gold standard for treatment. However, there has been known unresponsiveness to treatment, especially due to the involvement of other genes, such as the Janus kinase 2 (JAK2) gene. This study aimed to evaluate the relationships between JAK2 levels and complete hematological response (CHR), as well as early molecular response (EMR) after 3 months of imatinib treatment in patients with chronic phase CML. Patients with Ph+ CML in the chronic phase (n = 40; mean age, 40 ± 11 years) were recruited to complete assessments consisting of clinical examination and blood test, including evaluation of complete blood counts and the JAK2 levels, at baseline and following 3 months of therapy with imatinib (at an oral dose of 400 mg per day). Subjects were divided into two groups according to the presence of CHR and EMR. JAK2 gene levels, phosphorylated, and total JAK2 proteins at baseline were significantly lower in the group with the presence of CHR and EMR. In addition, baseline JAK2 levels, including JAK2 gene expression, phosphorylated, and total JAK2 proteins, were negatively correlated with the presence of CHR and EMR. Based on these findings, JAK2 levels may be a potential indicator for evaluating treatment response on imatinib due to its role in the pathophysiology of CML.

Effect of standardized Aronia Melanocarpa extract on oxidative stress and antioxidant status in patient with chronic myeloid leukemia treated with imatinib

Antioxidant status in patients with chronic myeloid leukemia (CML) is significantly decreased in comparison with healthy individuals. Oxidative stress (OS) may be associated with the pathophysiology of CML and can influence on development of resistance to imatinib. The aim of our study was to investigate the effect of Aronia melanocarpa extract (A-lixir 400 PROTECT®) on OS in CML patients treated with imatinib. In this study a total of 40 CML patients treated with imatinib for longer than 1 month were included: twenty patients were treated with imatinib and A-Lixir 400 PROTECT® (treatment group) and twenty patients were treated only with imatinib (control group). OS parameters (d-ROM, PAT and OSI) were measured at the initial visit, and after 21 and 42 days of treatment. Adjuvant treatment with A-Lixir 400 PROTECT® could lead to attenuation of OS. d-ROM and OSI in this group of patients were significantly higher at initial visit when compared to values after 21 and 42 days of treatment (p<0.05). Total antioxidant capacity (PAT) was significantly higher after 21 and 42 days of treatment initiation in comparison with the pretreatment values. In the control group no significant differences were obtained between investigated parameters at any time of measurement. We can conclude that adjuvant treatment with A-Lixir 400 PROTECT® after 21 and 42 days lead to significant reduction of OS in patients with CML treated with imatinib. Keywords: Oxidative stress, d-ROM, PAT, OSI, chronic myeloid leukemia, imatinib, Aronia melanocarpa

Chronic Myeloid Leukemia, from Pathophysiology to Treatment-Free Remission: A Narrative Literature Review.

Chronic myeloid leukemia (CML) is one of the most common leukemias occurring in the adult population. The course of CML is divided into three phases: the chronic phase, the acceleration phase, and the blast phase. Pathophysiology of CML revolves around Philadelphia chromosome that constitutively activate tyrosine kinase through BCR-ABL1 oncoprotein. In the era of tyrosine kinase inhibitors (TKIs), CML patients now have a similar life expectancy to people without CML, and it is now very rare for CML patients to progress to the blast phase. Only a small proportion of CML patients have resistance to TKI, caused by BCR-ABL1 point mutations. CML patients with TKI resistance should be treated with second or third generation TKI, depending on the BCR-ABL1 mutation. Recently, many studies have shown that it is possible for CML patients who achieve a long-term deep molecular response to stop TKIs treatment and maintain remission. This review aimed to provide an overview of CML, including its pathophysiology, clinical manifestations, the role of stem cells, CML treatments, and treatment-free remission.

Synthesis and biological evaluation of novel aromatic amide derivatives as potential BCR-ABL inhibitors

BCR-ABL1 kinase is a key driver of the pathophysiology of chronic myeloid leukemia (CML). Current treatments need to broaden the chemical diversity of BCR-ABL1 kinase inhibitors to overcome drug resistance. We designed and synthesized a series of aromatic amide derivatives based on several generations of BCR-ABL1 kinase inhibitors. Biological studies showed that compared with Imatinib, these compounds showed significant proliferation inhibitory activities of HL-60 and K562 in cell activity assay. Compounds 4g and 4j exhibited significant anti-tumor activity against the K562 cells with IC50 values of 6.03 ± 0.49 μM and 5.66 ± 2.06 μM respectively. Compounds 4g and 4j, as potential BCR-ABL1 inhibitors, inhibit the phosphorylation of ABL1 and CRKL in a dose-dependent manner. Therefore, compounds 4g and 4j can be used as a starting point for further optimization.

Demographic and Clinical Characteristics of Chronic Myeloid Leukemia Patients: A Study on Confined Populations of Southern India

Abstract Context: Chronic myeloid leukemia (CML) is one of the most common hematological malignancies in all populations throughout the world. Even though the pathophysiology of CML was well explained in majority of the studies, the incidence of CML was shown to exhibit population diversity, and hence, the demographic factors underlying CML origin remain to be understood. Further, the introduction of tyrosine kinase inhibitors had revolutionized the treatment of CML over the years; however, there is a need for developing tailoring therapy to individual risk since the patient clinical heterogeneity poses a major problem during drug response. Therefore, the study of basic clinical picture may aid in planning treatment strategies for CML patients. Aim: The aim of this article is to study the epidemiological and clinical variables associated with the prognosis of CML. Subjects and Methods: We have considered the distribution of various demographic and clinical variables among 476 CML patients diagnosed at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India. Statistical Analysis Used: All the analyses were performed through SPSS software (version 21.0). Correlation and Cox regression analyses were also performed. Results: Apart from the elevated male sex ratio in CML incidence, high frequency of males was observed to be nonresponders to imatinib mesylate (IM). IM response was shown to be dependent on phase of diagnosis, whereas overall survival of CML patients depends on the age at onset and response to IM. Conclusions: The study of epidemiology and clinical picture of CML patients may help in planning better treatment strategies at diagnosis to achieve long-term progression-free survival.

Modeling malignancies using induced pluripotent stem cells: from chronic myeloid leukemia to hereditary cancers

Over the last decade, the possibility of reprogramming malignant cells to a pluripotent state has been achieved in several hematological malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, and chronic myeloid leukemia (CML). It has been shown that it is readily possible to generate induced pluripotent stem cells (iPSCs) from several types of primary CML cells and to generate progenitors and differentiated cells with variable efficiency. Although these experiments have brought some new insights in the understanding of CML pathophysiology, the ultimate goal of generating induced leukemic stem cells (LSCs) with long-term multilineage potential has not yet been demonstrated. Experiments under way will determine whether additional signaling events are required to induce the emergence of bona fide LSCs. However, iPSC modeling offers the unique possibility to generate pluripotent cells harboring cancer-predisposing mutations using patient-derived noncancerous cells, as has been shown in Li-Fraumeni syndrome, BRCA-1 associated breast carcinomas, or RET-mutated medullary thyroid carcinomas. In these conditions, mutated iPSCs can then be used to study the mutational history that precedes the appearance of the malignant transformation and to develop novel drug-screening strategies. The ability to induce a successful differentiation program toward the tissue in which a given cancer develops or to generate tissue-specific cancer organoids in which the full oncogenic potential can be revealed remains a major challenge in the field. Similarly, in hematological malignancies, a significant hurdle remains due to the lack of adequate technology to induce the emergence of leukemic cells that resemble LSCs, which hinders our ability to study the mechanisms of therapy resistance.

Revisiting Early Stages of Chronic Myeloid Leukemia (CML): Evidence That Leukemic Progenitors Appear First in Peripheral Blood during Molecular Recurrence

The use of tyrosine kinase inhibitors (TKI) has dramatically changed the natural history and the prognosis of CML with a major improvement of survival in patients responding to therapy. However, tyrosine kinase inhibitor (TKI) discontinuation trials in patients with deep molecular responses (DMR), show that 50-60% of patients will have a molecular recurrence requiring to resume the TKI therapy. The great majority of these relapses occur during the first 6 months of TKI discontinuation. Although the sustained persistence of leukemic stem cells (LSC) is clearly the cause of the recurrent disease, cellular dynamics of these early relapses and the pattern of cells giving rise to them are not determined. We had the opportunity to study this pattern in 4 patients (pts) who presented molecular recurrence after Imatinib mesylate (IM) discontinuation (n= 3) or on TKI therapy with Dasatinib (n= 1) after obtaining a deep molecular response de grade MR 4.5. The total duration of TKI therapy before TKI discontinuation was 14 years (UPN1), 15 years (UPN2) and 30 months (UPN3). All three pts (Aged 47, 66, 87 respectively) had CML-CP treated with IM 400 mg as a first line therapy with induction of DMR in all three. The duration of DMR (MR4.5- was for 6, 5 and 2 years for UPN1, 2 and 3 respectively. The reasons for TKI discontinuation were the TKI-discontinuation protocol in French observatory (UPN1, UPN2) and suspicion of interstitial pneumonia (UPN3). The patient UPN4 was on Dasatinib as a second line therapy and in DMR for 3 years. At the time of detection of molecular recurrence, all 4 pts had normal clinical examination with normal complete blood counts (CBC) with no evidence of circulating immature blood cells. After IM-discontinuation, molecular relapses occurred at 2 months (BCR-ABL IS 27%) 3 months (BCR-ABL IS transcript level at 13.1%) and at 9 months (BCR-ABL IS transcript level at 2.7%.) in patients UPN1, 2 and 3 respectively. In UPN 4, the molecular recurrence (BCR-ABL IS transcript 10%) was documented at + 4 months. In all 4 patients, colony forming cell (CFC) assays were performed from the same blood samples in which the molecular recurrence was documented. At day + 14, clonogenic cells (CFU-GM, BFU-E, CFU-Mixed) were plucked and BCR-ABL expression in each individual or pooled (2-4) CFC was determined. For 5.104PBMC plated in methycellulose assays in culture, the mean numbers of CFC obtained varied between 12 and 29 CFC / dish. Despite the normal CBC, the presence of BCR-ABL-expressing CFC in blood was demonstrated in all 4 pts. In UPN1, all CFC (20/20) obtained from peripheral blood were positive for BCR-ABL with a heterogeneous expression amongst CFC (BCR-ABL /ABL ratio range 2- 25%, mean 33%). Interestingly in this patient, CFC and LTC-IC assays performed in a bone marrow aspirate prior to IM discontinuation showed no evidence of BCR-ABL-expressing progenitors, suggesting a rapid and massive expansion of a dormant LSC clone migrating to blood at the time of molecular relapse. In pt UPN2, 9 individual CFC were found to express BCR-ABL with a significant heterogeneity (BCR-ABL/ABL ratio 0.2% - 56% , mean = 15 %). In Pt UPN3, with the lowest levels of BCR-ABL during molecular relapse, 4/73 CFC expressed BCR-ABL. All three pts had a favorable response after resuming Imatinib (UPN1, 2, 3) or after switching therapy to Bosutinib (UPN4). On UPN4, 2 pools of 4 CFC/20 expressed BCR-ABL. In two pts with 100% clonogenic relapse (UPN 1 and UPN2) the estimated circulating leukemic CFC numbers were 607 / ml and 468 / ml of blood respectively, in the presence of normal appearing CBC. In UPN 3 and 4, with approximately 10% of CFC being BCR-ABL +, these numbers were estimated to be 507 and 45 per ml of blood, respectively. Thus, the study of these four patients demonstrates that circulating leukemic CFC are the first detectable abnormal cellular components during molecular recurrence and that the clonogenic relapse in peripheral blood precedes myeloid expansion. These results show also for the first time to our knowledge, that leukemic CFC egress from bone marrow to peripheral blood is an early event during early stages of CML and the CFC expansion takes place not only in bone marrow but also in peripheral blood. Besides shedding light into the pathophysiology of CML, these findings could be of major interest to predict relapse parameters and could lead to the design of novel biological follow-up protocols after TKI discontinuation. DisclosuresNo relevant conflicts of interest to declare.

Role of transcription factor C/EBPβ in the pathophysiology of chronic myeloid leukemia and as a potential target in eradicating leukemic stem cells with interferon-α

Tyrosine kinase inhibitors (TKIs) have markedly improved the prognosis of patients with chronic-phase chronic myeloid leukemia (CML). However, the development of a novel therapeutic strategy that can target and eradicate CML stem cells remains an important requirement to achieve a complete cure for CML because these stem cells cause relapse and drug resistance even in patients receiving TKI treatment. Interferon-α (IFNα) has long been used for the treatment of chronic-phase CML, and its efficacy is now being re-evaluated as a therapeutic option. Some clinical studies have demonstrated the additive efficacy of IFNα in CML patients treated with TKI. However, the mechanism of action is not fully understood. In this review, we introduce our recent findings on the effects of IFNα on CML stem cells via the transcription factor CCAAT/enhancer binding protein β, which regulates stress-induced hematopoiesis.

ETS1 Expression Is Upregulated in Chronic Myeloid Leukemia (CML) and the ETS1 Transcriptional Program Correlates with Disease Progression Towards Blast Crisis

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value<0.0001). Among this ETS1 program specific of BC, we identified 2 targets that are discriminant for CD34+ cells of CML patients without cytogenetic response under Imatinib therapy. These two genes were Dynamin 3 (DNM3) and LIMS1/ PINCH1. Dynamin 3 is a member of the motor proteins implicated in cell motility and cytokinesis and it is implicated in megakaryocyte development whereas LIMS1 / PINCH1 is essentially involved in cell migration and adhesion. In order to validate ETS1 and its target expressions in a novel cohort of CML patients, we have analyzed the expression of DNM3 and PINCH1 in whole blood samples in CML patients ( n= 60 ) as compared to healthy controls (n= 32). We have found that, similar to ETS1, DNM3 and LIMS1 / PINCH1 mRNA were highly upregulated in primary CML and this increase was highly significant. In this new cohort of patients, DNM3 transcript levels were inversely correlated to WBC count (r=-0.32, p-value=0.011) and especially in male gender (r=-0.43, p-value=0.012). The expression of LIMS1 was inversely correlated with the age at diagnosis and the major molecular response at 12 months. Thus, our results show for the first time the major upregulation of ETS1 transcriptional program in CML. ETS1 transcriptional program has been found to correlate with the gene expression pattern involved in the progression of the disease in the transcriptomic analysis of CML CD34+ cells. The novel druggable targets that we identified in the BCR-ABL-activated transcriptional program is currently under investigation to determine their use in patients with resistance to targeted therapies. DisclosuresNo relevant conflicts of interest to declare.

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

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. DisclosuresTurhan:Bristol Myers Squibb: Consultancy; Novartis: Research Funding.

The alteration and clinical significance of Th22/Th17/Th1 cells in patients with chronic myeloid leukemia.

T helper- (Th-) cell immunodeficiency plays important roles in tumor development and their effects in chronic myeloid leukemia (CML) remain unclear. In the present study, we mainly investigated the role of Th22, Th17, and Th1 cell and their related cytokines (IL-22, IL-17, and IFN-r) in the pathophysiology of CML. Bone marrow (BM) and peripheral blood (PB) were extracted from newly diagnosed (ND), chronic phase- (CP-) CML patients, and controls. Th subsets were examined by flow cytometry. Plasma IL-22, IL-17, and IFN-r concentrations were measured by ELISA. AHR and RORC mRNA expressions were examined by RT-PCR. The frequencies of Th22, Th17, and Th1 cells, along with the expression of specific transcription factors RORC and AHR, were significantly decreased in ND patients compared with healthy controls, while all these abnormality recovered in CP patients. In addition, there existed a significantly positive relationship between Th22 and Th17 cells in PB or BM. A significantly negative relationship was found between Th cells (Th22, Th17, or Th1) and BCR-ABL (%) IS or the number of PB white blood cells. All these results demonstrated that Th22, Th17, and Th1 cells might be important therapeutic targets in CML and could facilitate a better outcome for tumor immunotherapy.

Insights into the pathophysiology and therapy of myeloproliferative neoplasms from mouse models.

CML (chronic myeloid leukemia) is genetically simple cancer, having a very few point mutations or copy number variations in its early chronic phase genome. Therapies using ABL1 tyrosine kinase inhibitors (TKIs) have dramatically altered the prognosis in CML. The major goal of current molecular research on CML is to understand TKI resistance and disease progression. CML mouse models can provide information and insights into the pathophysiology of this disease that are difficult or impossible to obtain when one is limited to working with human cells. There are two widely used mouse models of CML: binary conditional BCR-ABL1 transgenic mice1, 2 and the retroviral bone marrow transplant (BMT) model of CML.3, 4, 5 In these models, BCR-ABL1-induced CML-like myeloproliferative neoplasm originates from stem/progenitor cells with multi-lineage repopulating activity.5 This neoplasm is transplantable and can progress to blast crisis;5, 6 it is responsive to kinase inhibitors and immunological therapy.7, 8 In this summary, I will focus on three examples where mouse model systems have revealed crucial details about the pathophysiology of CML.

Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy

Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of the role of such tyrosine kinases in these diseases has led to the development of improved therapies with tyrosine kinase-targeted inhibitors. However, most drugs, including new KIT D816V-blocking agents, have failed to achieve long-lasting remissions in advanced systemic mastocytosis, and there is a similar problem in chronic myeloid leukemia, where imatinib-resistant patients sometimes fail to achieve remission, even with second- or third-line BCR-ABL1 specific tyrosine kinase inhibitors. During disease progression, additional signaling pathways become activated in neoplastic cells, but most converge into major downstream networks. Among these, the AKT and STAT5 pathways appear most critical and may result in drug-resistant chronic myeloid leukemia and systemic mastocytosis. Inhibition of phosphorylation of these targets has proven their crucial role in disease-evolution in both malignancies. Together, these observations suggest that STAT5 and AKT are key drivers of oncogenesis in drug-resistant forms of the diseases, and that targeting STAT5 and AKT might be an interesting approach in these malignancies. The present article provides an overview of our current knowledge about the critical role of AKT and STAT5 in the pathophysiology of chronic myeloid leukemia and systemic mastocytosis and on their potential value as therapeutic targets in these neoplasms.

Superoxide Dismutase 2 (Sod2) Expression Is Highly Decreased In Chronic Myeloid Leukemia (CML): Contribution To Genetic Instability In Bcr-Abl-Expressing Leukemic Cells

A increased oxidative stress is one of the hallmarks of chronic myeloid leukemia (CML). Manganese Superoxide dismutase 2 (SOD2) is a key scavenger enzyme against reactive oxygen species (ROS) in eukaryotic cells, playing a crucial role in antioxidant defense but its potential implication in the CML pathophysiology is not determined. We have analyzed the expression of SOD2 at the RNA level in a large cohort of CML patients (n=49) compared to a cohort of controls with non-malignant hyperleucocytosis (n= 19). This analysis showed a major decrease of SOD2 mRNA, inversely correlated with leucocytosis (p< 0.0001) and with Sokal Score, high-risk patients showing a lower expression of SOD2 (p= 0.019). To determine the effects of the silencing of SOD2 gene in an experimental CML model, we analyzed SOD2 expression in the human UT7 cell line expressing either native or BCR-ABL carrying T315I mutation. SOD2 protein levels in BCR-ABL expressing cells were similar to parental UT7 cells, suggesting that BCR-ABL has no direct effect on SOD2 expression. We have then silenced SOD2 expression in UT7 cells as well as UT7-BCR-ABL cells. Interestingly, the expression of H2O2 levels measured with flow cytometry was not changed after SOD2 inhibition, suggesting activation of compensatory anti-oxidant pathways. A global analysis of 27 anti-oxidant gene expression showed a reduction of PRDX2 only in BCR-ABL-expressing cells. To analyze same cells at the genomic level, we have performed CGH arrays in SOD2 silenced UT7, UT7-BCR-ABL and UT7-T315I cells as compared to their counterparts expressing SOD2. These experiments showed a major genetic instability with deletions and duplications in several chromosomal loci only in native or T315I-mutated BCR-ABL expressing cells silenced for SOD2. The 13q31.3-q34 locus, containing GPC5 and GPC6 genes, was found to be duplicated in non-mutated BCR-ABL cells deficient in SOD2 expression. GPC5 and GPC5 belong to the Glypican family, which are cell surface heparan sulfate proteoglycans anchored to the cell membrane via glycosyl-phosphatidyl-inositol-structures. The expression of several GPC is increased in some malignancies, including glioma, pancreatic carcinoma, breast cancer (GPC1), hepatocellular carcinoma (GPC3) and rhabdomyosarcoma (GPC5). Furthermore, the 13.q31.3 region, duplicated in BCR-ABL-expressing cells upon SOD2 deletion, contains the polycistronic mir-17-92 cluster (mir-17, mir-18a, mir19a, mir-20a, mir-19b1, mir-92a1), which is highly increased in CML CD34+ cells. Interestingly, the inhibition of SOD2 expression did not lead to the same genomic modifications detected by CGH arrays; the duplication of the 13q31.3 and 13q31.3q24 regions was not found in UT7 BCR-ABL cells harboring the T315I mutation. T315I cells silenced for SOD2 expression exhibited multiple abnormalities among which a large deletion of the 20q11.21 region which encodes the defensin gene family (DEFB115, DEFB116, DEFB118, DEFB119, DEFB121, DEFB122, DEFB123, DEFB124; REM1, DUSP15). Beta-defensins are antimicrobial peptides, which are part of the innate immunity against pathogens. The loss of beta-defensin 1 gene has already been reported in solid tumors. In conclusion, we demonstrate in a large cohort of CML patients, a major decrease of SOD2 expression, involved in antioxidant defense. We further show for the first time a link between loss of SOD2 expression and genetic instability in BCR-ABL expressing cells as demonstrated by CGH arrays. Genetic consequences of reduced SOD2 expression in CML should be further analyzed in prospective studies as patients with low SOD2 expression could be more prone to develop a mutator phenotype under TKI therapies. Disclosures:Guilhot:Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:Bristol Myers Squibb, Novartis: Consultancy, Honoraria.

BRCA1-Down-Regulation In Chronic Myeloid Leukemia (CML) Occurs Via The Deubiquitinase / Tumor Suppressor BRCA1-Associated Protein 1 (BAP1)

BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation. Disclosures:Guilhot:Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.

The Alteration of SEPT5 Gene Expression in BCR-ABL Positive Cells and Its Possible Correlation with the Development and / or Progression of Chronic Myeloid Leukemia (CML)

Abstract 4415The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett's test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p <0.05). The spontaneous and stimulated with IL-8 chemotaxis assays used granulocytes and were assessed using chamber containing 96 wells. However, although the results suggest an increased chemotactic activity in patients, there were no significant differences (p<0.05) between controls and patients – regardless of whether the chemotaxis was spontaneous or stimulated with IL-8. In mammals the SEPT5 gene is associated with cellular processes such as exocytosis, apoptosis, leukemogenesis, carcinogenesis and neurodegeneration. Therefore, molecules capable of interacting with the septins, either at biochemical or molecular level, can bring information about their functions in cytokinesis. Studies indicate that the human septins can interact among themselves and with other components of the cytoskeleton – this may be a relevant observation regarding the function of this gene in cancer. The SEPT5 can be activated by different pathways – this may increase expression in translocated cells. Despite major advances in the treatment of CML, the treatments available are not capable of inactivating all the signaling pathways activated by BCR/ABL. Our results demonstrate that SEPT5 may be involved in the pathophysiology of CML. Also, it is clear the importance of the study of pathways that could culminate in its high expression or the triggering of other unknown pathways involved in the development of CML. The increased expression of this gene may be related to disease progression, and finally, the identification of several important genes may lead to a better understanding of CML and helping to identify new therapeutic targets. FAPESP/INCT. Disclosures:No relevant conflicts of interest to declare.

Distinct Functions of Stat5A and Stat5B in Chronic Myeloid Leukemia (CML): Stat5B Is Implicated in Survival and Self-Renewal and Stat5A in Imatinib Resistance.

AbstractAbstract 1214The use of tyrosine kinase inhibitors (TKI) has dramatically changed the prognosis and the natural history of CML. However the occurrence of drug resistances, especially at the stem cell level, requires the identification of novel functional targets. The signal transducer and activator of transcription 5 (Stat5), constitutively phosphorylated by BCR-ABL, is thought to be involved in the pathophysiology of CML, especially by its contribution to the antiapoptotic phenotype observed in CML cells via activation of Bcl-xL. There is currently no data linking clearly the involvement of Stat5 activation to TKI resistance in CML. Moreover, it has not been established, if the two closely related Stat5 factors, Stat5A and Stat5B, fulfill the same or distinct roles in leukemic cells. In order to study these potentially differential functions, we have specifically inhibited Stat5A and Stat5B through lentivirus-mediated RNA interference in human Ph+ CML cell lines K562, LAMA84 and Meg01 and their Imatinib mesylate (IM)-resistant counterparts. We have also transduced with the same lentiviral vectors, primary human CML CD34+ cells and evaluated the effects of Stat5A or B invalidation in terms of hematopoietic stem cell self-renewal in long-term culture initiating cell (LTC-IC) assays. In preliminary experiments, the successful inhibition of Stat5A or B was demonstrated using Western blotting with specific inhibition of each factor with the corresponding shRNA. In CML cell lines, Stat5B inhibition led to a massive apoptosis with growth arrest in all three cell lines whereas Stat5A inhibition had no effect. Surprisingly, Stat5B was active in the absence of canonical tyrosine phosphorylation and its absence strongly enhanced the apoptosis-inducing effects of TKI (Imatinib and Nilotinib). We then evaluated the role of Stat5A or B in the context of CML cell lines resistant to IM, in which there was no detectable ABL-kinase domain mutations neither MDR amplification. In this context, the Stat5A and B protein levels were identical but there was a strong over-activation of Stat5, as detected by Tyr694/699 phosphorylation status. In the two resistant sublines LAMA84-R and Meg01-R, anti-Stat5A and anti Stat5B selective immunoprecipitation assays further revealed that only Stat5A, and not Stat5B, was Tyr-phosphorylated. Therefore, in two independent IM-resistant sublines, LAMA84-R and Meg01-R, resistance correlated with over-activation of Stat5A, in the absence of canonical activation of Stat5B.We next investigated the role of Stat5A or B in primary CML stem cell self-renewal, using CD34+ cells from CML patients in chronic phase (CP-CML) at diagnosis. CD34+ cells were transduced with control (luciferase)-, Stat5A- or Stat5B-shRNA encoding lentiviral vectors and Long Term Colony Initiating Cell (LTC-IC) assays were performed. In four out of five patient samples, Stat5B-shRNA induced a massive inhibition of the LTC-IC activity. Stat5A-shRNA, on the reverse, had highly variable impact depending on the patients, either enhancing or somehow decreasing LTC-IC activity. We also performed similar assays with CD34+ progenitor cells collected from healthy donors. Except for one sample, Stat5B knock down had rather weak consequences on LTC-IC activity. Finally, to extend the potential roles of Stat5A and B in Imatinib resistance, we have transduced CD34+ cells of an IM-resistant CML patient whose cells had also been shown to be resistant to IM in vitro. GFP+ Stat5A-shRNA transduced cells were cell-sorted by FACS and their IM-resistance was tested in clonogenic assays. As compared to the control Luciferase shRNA vector, Stat5A inactivation reduced the colony numbers by 75%, with development of small sized colonies.Thus, these data strongly suggest that Stat5b plays a major role in the survival of CML cells, especially in the primitive hematopoietic cells, extending the results obtained in CML cell lines. Our data further show for the first time that this effect takes place in the absence of Tyrosine phosphorylation of Stat5B. Conversely, CML cells express tyrosine-phosphorylated Stat5A, whose levels strongly increased in TKI-resistant cells and its inhibition by an shRNA strategy leads to the reversal of TKI resistance of phenotype. Thus, Stat5A and Stat5B exhibit entirely distinct - not interchangeable- oncogenic activities in CML pathophysiology and represent novel therapeutic targets. Disclosures:No relevant conflicts of interest to declare.

Oxidative stress and antioxidant status in patients with chronic myeloid leukemia.

Chronic myeloid leukemia is a myeloproliferative disorder with a unique rearrangement, the Philadelphia chromosome. Oxidative stress, a pervasive condition of an increased number of reactive oxygen species, is now recognized to be prominent feature of various diseases and their progression. Thus antioxidants, which control the oxidative stress state, represent a major line of defense regulating overall true state of health. The relationship between antioxidants status and levels of well-known markers of oxidative stress that are measured as lipid peroxides and oxidized proteins reflect better health indices and postures. The aim of this study was to evaluate the role of oxidative stress in pathophysiology of Chronic myeloid leukemia by measuring the circulating plasma lipid peroxide levels in terms of malonyldialdehyde, total lipid hydroperoxide and oxidized proteins as protein carbonyl whereas antioxidant status were estimated in terms of reduced glutathione and total thiol in plasma of Chronic myeloid leukemia patients. The present study included 47 Chronic myeloid leukemia patients and 20 age-and sex-matched healthy subjects. Out of 47 Chronic myeloid leukemia patients, 31 were in chronic phase (CML-CP) and 16 in accelerated phase (CML-AP). The median age of Chronic myeloid leukemia patients was 33 years and that of controls was 32 years. Oxidative stress and antioxidant status in plasma were evaluated by spectrophotometric procedures. There was a significant increase (p<0.05) in plasma malonyldialdehyde, total lipid hydroperoxide and protein carbonyl levels in Chronic myeloid leukemia patients as compared to healthy subjects. Our results also showed that plasma malonyldialdehyde and protein carbonyl levels were markedly elevated (p<0.05) in both chronic phase (CML-CP) and accelerated phase (CML-AP) as compared to healthy volunteers. Antioxidant status was found to be significantly decreased (p<0.05) in Chronic myeloid leukemia patients and its phases as compared to healthy participants. It could be concluded that oxidative stress may be associated with the pathophysiology of Chronic myeloid leukemia.

Novel Abl Kinase Inhibitors in Chronic Myeloid Leukemia in Blastic Phase and Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome, which is associated with a balanced translocation involving chromosomes 9 and 22 to produce a fusion gene (bcr-abl) that gives rise to a constitutively activated Abl tyrosine kinase. This kinase led to the discovery of several small-molecule inhibitors, imatinib being the first and most successful of these. Resistance to imatinib results in some patients from Abl kinase point mutations. Overcoming imatinib resistance represents one of the biggest challenges facing clinicians in the modern management of CML. In this review, we discuss the current understanding of CML pathophysiology and mechanisms of imatinib resistance and how advancing this knowledge has led to the design of novel therapies in the area of blastic phase CML and Philadelphia chromosome–positive acute lymphoblastic leukemia with previous imatinib failure.