Chronic Myeloid Leukemia Samples Research Articles

Variant-specific BCR::ABL1 quantification discrepancy in chronic myeloid leukemia.

Accurate quantification of the BCR::ABL1 fusion gene in whole blood is pivotal for the clinical management of chronic myeloid leukemia (CML) patients. The fusion protein encoded by BCR::ABL1 can vary in size, depending on the BCR and/or ABL1 gene breakpoint. The vast majority of CML patients have a p210 BCR::ABL1 fusion gene (M-BCR), which can be attributed to the presence of either e14a2 (b3a2) or e13a2 (b2a2) mRNA transcript junctions. Twenty-five CML samples were analyzed in two different ISO15189-accredited centers that both use an Europe Against Cancer-based quantitative polymerase chain reaction (qPCR) protocol. Reanalysis of the sample set with transcript-specific standard curves and digital droplet PCR (ddPCR) were performed. qPCR quantification revealed a significant (up to 1 log) difference specifically for the e13a2 transcript variant in contrast to e14a2 transcripts (Hodges-Lehman 4.29; p < 0.001). Reanalysis of the sample set with transcript-specific standard curves abolishes the initial transcript-specific difference (Hodges-Lehman 0.003; p = 0.8192). Comparison of transcript-specific qPCR results of both centers with ddPCR, an absolute quantification method, showed a statically significant association, especially in the lower range, indicating the clinical utility of transcript-specific or absolute quantification methods. Our data show that differences between transcript-specific quantification might exist between centers, leading to potential clinical impact on the follow-up of CML patients. The use of transcript-specific standard curves for qPCR quantification, or absolute quantification, can significantly reduce these differences. Specific attention should be applied to the interpretation of quantification differences of CML patients that switch between diagnostic centers.

Mutations in myeloid transcription factors and activated signaling genes predict chronic myeloid leukemia outcomes

AbstractAdvancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) toward precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifier genes and activated signaling/myeloid transcription factors (AS/MTFs) with disease progression and treatment failure in patients with CML after tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)–based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes, with a limit of detection of 0.2%. Seventy mutations were detected in 57 diagnostic samples (22.4%), whereas 64 mutations were detected in 39 of the follow-up samples (27.9%). Carrying any mutation at initial diagnosis was associated with worse outcomes after TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with imatinib showed higher risks of treatment failure (hazard ratio, 2.53; 95% confidence interval, 1.13-5.66; P = .0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs. The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival, and progression risk. Additionally, there was an observable nonsignificant trend indicating a heightened risk of progression to advanced disease and worse overall survival. In conclusion, mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression and may help upfront TKI selection.

Uncovering the Transcriptional Landscape of CML Development and Therapeutic Interventions Via State-Transition Modeling

Chronic Myeloid Leukemia (CML) is driven by expression of the BCR-ABL fusion gene. Despite the success of treatment with tyrosine kinase inhibitors (TKIs), the disease-originating leukemic stem cells frequently persist through therapy, and disease cannot be eradicated. Thus, new biomarkers beyond BCR/ABL may be necessary to assess disease state and treatment response, and to identify novel targets and therapeutics that could lead to a cure. Herein, we used time-series RNA-seq to molecularly characterize the transcriptional states of CML and predict disease evolution and treatment response. Using weekly blood draws from a murine tetracycline (Tet)-inducible BCR/ABL expression model of chronic phase CML and non-BCR/ABL expressing controls ( Fig 1A), we applied state-transition theory to mathematically model CML development. We constructed a CML state-space using dimensionality reduction to the full transcriptomic data of the time-series CML and control mice. From the sample distribution in the state-space, we empirically derived healthy (no BCR/ABL) and perturbed (BCR/ABL-induced) potentials ( Fig 1B). Using these potentials, our model described the transcriptome evolution during CML development as a particle undergoing Brownian motion. We used the Fokker-Planck (FP) solution of the Langevin equation of motion to both accurately model the transcriptome and predict the future dynamics from a single sample used as an initial condition. While control mice remain in the region of the state-space corresponding to health, each of the BCR/ABL expressing mice moved toward CML state as early as one week after BCR/ABL induction and before detection of BCR/ABL mRNA expression. Of note, no significant difference was observed between the experimental and the FP predicted trajectories and survivals, supporting the accuracy of our approach in predicting disease evolution and outcome at the earliest time points. The CML potential was described as a three well potential with three stable (Early c1, Transition c3, Late c5) and two unstable (Early Transition c2 and Late Transition c4). Differentially expressed genes (DEGs) corresponding to Early c1, Transition c3, Late c5 states were identified by comparing with control mice. Importantly, the state-space model also allowed assessment of whether each DEG had a pro- or anti-CML effect. At the Early state c1, we identified 78 DEGs; 55% of them were anti-CML, indicating an early attempt of the biological system to prevent disease development. By GSEA, we showed that the anti-CML Hallmark gene sets included EMT, angiogenesis, hypoxia and the only pro-CML gene set was IL2/Stat5. In contrast, at the Transition c3 and Late c5 states, we identify 366 and 1858 DEGs, respectively; nearly all these DEGs had a pro-CML effect and were enriched for Hallmark gene sets involving inflammation, angiogenesis and metabolism. Using CML state-space as a pseudotime ordering of the CML samples, we also studied modules of non-linear gene expression dynamics. This approach revealed changes in gene expression rates (inflection points) coinciding with the unstable critical points (c2 and c4) that separated the otherwise stable states. The genes in these transition modules included immune/inflammation processes and likely represented those genes that drove CML state-transition. To investigate the evolution of disease “in reverse” (i.e., after the CML phenotype developed), we treated two cohorts of CML mice, one with a TKI (nilotinib) for 4 weeks and one with Tet to turn off the expression of BCR/ABL (Tet-off, Tet-on [TOTO]). We adapted the state-transition model to a “treatment state-transition” model by introducing a specific parameter, g, which both accurately described and predicted individual treatment efficacy. We showed that although the circulating leukemic cells were suppressed by TKI, the disease state recovered only from c5 to c3, but never reached the health (control) state, and the treated mice rapidly relapsed once treatment was completed. Conversely, the TOTO cohort, which had the BCR/ABL expression completed turn off, returned stably to the healthy state. In conclusion, our state-transition model provides a theory-based analytic framework for investigating disease dynamics and predicting treatment response and outcome. Importantly, can be translated into the clinic for novel transcriptomic-based diagnostic approaches.

Histone demethylase PHF8 facilitates the development of chronic myeloid leukaemia by directly targeting BCR::ABL1.

Although tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukaemia (CML), TKI resistance remains a major challenge. Here, we demonstrated that plant homeodomain finger protein 8 (PHF8), a histone demethylase was aberrantly enriched in CML samples compared to healthy controls. PHF8 inhibited CML cell differentiation and promoted CML cell proliferation. Furthermore, the proliferation-inhibited function of PHF8-knockdown have stronger effect on imatinib mesylate (IM)-resistant CML cells. Mechanistically, we identified that PHF8 as a transcriptional modulator interacted with the promoter of the BCR::ABL1 fusion gene and alters the methylation levels of H3K9me1, H3K9me2 and H3K27me1, thereby promoting BCR::ABL1 transcription. Overall, our study suggests that targeting PHF8, which directly regulates BCR::ABL1 expression, is a useful therapeutic approach for CML.

Molecular subtypes predict therapeutic responses and identifying and validating diagnostic signatures based on machine learning in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematological tumor derived from hematopoietic stem cells. The aim of this study is to analyze the biological characteristics and identify the diagnostic markers of CML. We obtained the expression profiles from the Gene Expression Omnibus (GEO) database and identified 210 differentially expressed genes (DEGs) between CML and normal samples. These DEGs are mainly enriched in immune-related pathways such as Th1 and Th2 cell differentiation, primary immunodeficiency, T cell receptor signaling pathway, antigen processing and presentation pathways. Based on these DEGs, we identified two molecular subtypes using a consensus clustering algorithm. Cluster A was an immunosuppressive phenotype with reduced immune cell infiltration and significant activation of metabolism-related pathways such as reactive oxygen species, glycolysis and mTORC1; Cluster B was an immune activating phenotype with increased infiltration of CD4 + and CD8 + T cells and NK cells, and increased activation of signaling pathways such as interferon gamma (IFN-γ) response, IL6-JAK-STAT3 and inflammatory response. Drug prediction results showed that patients in Cluster B had a higher therapeutic response to anti-PD-1 and anti-CTLA4 and were more sensitive to imatinib, nilotinib and dasatinib. Support Vector Machine Recursive Feature Elimination (SVM-RFE), Least Absolute Shrinkage Selection Operator (LASSO) and Random Forest (RF) algorithms identified 4 CML diagnostic genes (HDC, SMPDL3A, IRF4 and AQP3), and the risk score model constructed by these genes improved the diagnostic accuracy. We further validated the diagnostic value of the 4 genes and the risk score model in a clinical cohort, and the risk score can be used in the differential diagnosis of CML and other hematological malignancies. The risk score can also be used to identify molecular subtypes and predict response to imatinib treatment. These results reveal the characteristics of immunosuppression and metabolic reprogramming in CML patients, and the identification of molecular subtypes and biomarkers provides new ideas and insights for the clinical diagnosis and treatment.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

Intracellular angiopoietin-1 promotes TKI-resistance via activation of JAK/STAT5 pathway in chronic myeloid leukemia.

Drug resistance from BCR-ABL tyrosine kinase inhibitors (TKIs) and other chemotherapeutics results in treatment failure and disease progression in chronic myeloid leukemia (CML). However, the mechanism is still uncertain. In this study, we investigated the role of angiopoietin-1 (ANG-1) as a potential prognostic factor for drug resistance in CML. Both intracellular and secretory ANG-1 (iANG-1 and sANG-1) were overexpressed in multidrug-resistant CML samples. The IC50 value was higher in primary CD34+ CD38- cells with more ANG-1. Silencing ANG-1significantly sensitized three TKI-resistant CML cell lines to imatinib (IM) while recombinant human ANG-1 failed to retain cell survival in vitro. This indicated the important role of iANG-1 as opposed to sANG-1 in CML drug resistance. Moreover, a similar effect was observed in xenograft mice models bearing ANG-1-silenced CML cells. Subsequently, pathway analysis and protein validation experiments showed activation of the JAK/STAT pathway and augmentation of STAT5a phosphorylation in ANG-1 restored CML cells. Upstream Src phosphorylation, which plays a crucial role in CML drug resistance, was also upregulated as a key event in iANG-1-related JAK/STAT pathway activation. In conclusion, our study elucidated a new BCR-ABL independent molecular mechanism induced by intracytoplasmic ANG-1 overexpression as a potential strategy for overcoming CML resistance.

Integrated High-Throughput Drug Profiling and CRISPR Screening Identify Novel Pathway Vulnerabilities of Leukemic Stem Cells in Chronic Myeloid Leukemia

Despite BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) have dramatically improved the management of chronic myeloid leukemia (CML) patients, TKIs fail to eradicate leukemic stem cells (LSCs). CML-LSCs are recognized as BCR-ABL-kinase activity independent and responsible for TKI-resistance and relapse upon treatment discontinuation. Systematic mapping of druggable signaling pathways enables the identification of novel CML-LSCs effectively targeting drugs, which is critical to enhance the achievement of sustained treatment free remission in CML patients. We implemented ex-vivo drug testing of 80 targeted drugs and 18 drug combinations across 10 000-fold concentration, to evaluate drug sensitivity profiles of primary CML-LSCs and progenitors (CD34+ cells) from twenty patients in addition to CD34+ cells from three healthy donors. We identified novel drug vulnerabilities of CML-LSCs including VEGFR-, CDK2/WEE-, MDM2-, and PI3K/mTOR- inhibitors. Combination of imatinib with idasanutlin, venetoclax, AZD1775 and dexamethasone led to synergistic inhibition of CML-LSCs. To delineate the phenotype-specific sensitivity profile of CML CD34+ cells, we applied ex-vivo flowcytometry-based drug screening approach to bone marrow samples from ten CML patients using a custom library of 20 drugs. We identified specific sensitivity of CML CD34+CD38- cells to mepacrine, RG7112, idasanutlin, and navitoclax. In particular, mepacrine showed striking ability to induce differentiation of CML-LSCs, which was further validated in a separate cohort of primary CML samples and cell lines. To characterize the mechanisms of action of the discovered drugs, we applied genome-scale CRISPR screening in CML cells to identify potential sensitizing and resistance targets for five selected drugs. Imatinib, dasatinib, and tivozanib showed a similar profile of resistance and sensitizing genes, while AZD1775 and mepacrine had unique profiles. We identified knockout targets that confer BCR-ABL1-independent resistance common to different TKIs including mediator complex (MED23, MED26, CCNC), and apoptosis related (APAF1) genes in addition to other genes with uncharacterized role in CML, such as KCTD5 and EIF2AK1. On the other hand, knockout of certain genes was associated with increased sensitivity to specific drugs such as IGF1R-dasatinib and SPNS1-mepacrine associations. KCTD5 was consistently identified as the top resistance gene to all tested TKIs, and therefore we next investigated the potential role of KCTD5 in CML pathogenesis and TKI resistance. In RNA sequencing data, KCTD5 was significantly downregulated in CML-CD34+ cells compared to healthy counterparts. Using co-immunoprecipitation, we revealed that KCTD5 directly interacts with BCR-ABL and mediate TKI-induced ubiquitination by E3 ligase, as shown by WB assay, that can destine BCR-ABL for subsequent degradation. Knockout of KCTD5 was associated with decreased ubiquitination of BCR-ABL and resistance to imatinib in CML cell lines and primary CML-CD34+ cells. Furthermore, combination of TKI with deubiquitinase inhibitors induced enhanced TKI sensitivity in KCTD5-KO cells. In conclusion, we discovered novel mechanisms of primary TKI-resistance and drug vulnerabilities of CML-LSCs, which potentially enable eradication of LSCs in CML patients and hence pave the way for the achievement of functional cure in CML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression of deubiquitinase genes and inflammatory response in myeloid leukemia

Myeloid leukemia (ML) is a cancer of the blood that begins when cells of the myeloid lineage uncontrollably change and grow. Acute myeloid leukemia (AML) is a disorder of rapid, uncontrolled growth of immature myeloid cells in the blood and bone marrow. Chronic myeloid leukemia (CML) is characterized by the aberrant proliferation of myeloid cells and driven by the translocation of regions of the BCR and ABL genes to form the Philadelphia (Ph) chromosome. The deubiquitinase enzymes (DUBs) including A20, OTUB1, OTUB2, and Cezanne play important roles in inhibiting NF-κB activation in response to various stimuli. Cytokines including tumor necrosis factor-alpha (TNF-α), IL-6, and IL-1β are released from immune cell activation triggered by antigenic stimulation. To this end, blood samples of 20 AML and 62 CML patients and the control group consisting of 37 healthy individuals were used to examine the mRNA expression of A20, OTUB1, OTUB2 and Cezanne genes by using quantitative RT-PCR and determine IL-6, TNF-α and IL-1β concentrations by using ELISA. As a result, the mRNA level of OTUB1 was significantly decreased in both AML and CML patients compared to that in healthy individuals, however, no difference in the transcriptional expression of OTUB2 among AML and CML patients and control group was detected. Unlike the levels of OTUB1 and OTUB2, the expressions of A20 and Cezanne in CML, but not in AML patients were significantly lower than healthy individuals. For serum cytokine analysis of the study groups, in AML and CML samples, IL-6 and TNF-α concentrations significantly increased in comparison with the control group, however, IL-1β level was similar among CML, AML patients and healthy individuals. In conclusion, this study revealed the different DUB involvement in the pathogenesis of ML, suggesting further investigations on gene polymorphisms and their functions linked to biological properties of leukemia cells.

RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia.

The Philadelphia 9;22 chromosome translocation has two common isoforms that are preferentially associated with distinct subtypes of leukemia. The p210 variant is the hallmark of chronic myeloid leukemia (CML) whereas p190 is frequently associated with B-cell acute lymphoblastic leukemia. The only sequence difference between the two isoforms is the guanidine exchange factor domain. This guanidine exchange factor is reported to activate RHO family GTPases in response to diverse extracellular stimuli. It is not clear whether and, if so, how RHOA contributes to progression of p210 CML. Here we show that knockout of RHOA in the K562 and KU812, p210-expressing cell lines leads to suppression of leukemogenesis in animal models in vivo. RNA-sequencing analysis of the mock control and null cells demonstrated a distinct change in the gene expression profile as a result of RHOA deletion, with significant downregulation of genes involved in cell activation and cell adhesion. Cellular analysis revealed that RHOA knockout leads to impaired cell adhesion and migration and, most importantly, the homing ability of leukemia cells to the bone marrow, which may be responsible for the attenuated leukemia progression. We also identified IGFBP2 as an important downstream target of RHOA. Further mechanistic investigation showed that RHOA activation leads to relocation of the serum response factor (SRF) into the nucleus, where it directly activates IGFBP2. Knockout of IGFBP2 in CML cells suppressed cell adhesion/invasion, as well as leukemogenesis in vivo. This elevated IGFBP2 expression was confirmed in primary CML samples. Thus, we demonstrate one mechanism whereby the RHOA-SRF-IGFBP2 signaling axis contributes to the development of leukemia in cells expressing the p210 BCR-ABL1 fusion kinase.

Successful Preservation of Native BCR::ABL1 in Chronic Myeloid Leukemia Primary Leukocytes Reveals a Reduced Kinase Activity.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the acquisition of t(9;22) generating the fusion tyrosine kinase BCR::ABL1. However, despite the crucial role of this protein in the dysregulation of numerous signal transduction pathways, a direct measure of BCR::ABL1 kinase activity in chronic phase (CP) CML was never accomplished due to intense degradative activity present in mature leukocytes. Therefore, we developed a procedure suitable to preserve BCR::ABL1 protein under non-denaturing, neutral pH conditions in primary, chronic phase (CP)-CML samples. As a result, specific kinase activity was detected utilizing a biotinylated peptide substrate highly selective for c-ABL1. Furthermore, through this approach, BCR::ABL1 kinase activity was barely detectable in CP-CML compared to Ph+ acute lymphoblastic leukemia primary samples, where kinase activity is comparable to those measured in Ph+ cell lines. These in vitro findings provide the first direct measure of BCR::ABL1 kinase activity in primary CP-CML and reveal the presence of a still uncharacterized inhibitory mechanism that maintains BCR::ABL1 in a low activity state in CP-CML despite its overexpression.

Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors.

Simple SummaryNeutrophil extracellular traps (NETs) are a recently described form of neutrophil cellular death that has been associated with a thrombotic tendency in many diseases. We studied NET formation in neutrophils derived from patients with chronic myeloid leukemia (CML) and in CML neutrophil cell lines and demonstrated that NETs are increased in CML and that certain drugs used to treat CML (tyrosine kinase inhibitors—TKIs) increase NET formation. These findings may shed light on a novel mechanism linking CML, TKIs and vascular toxicity.Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.

Gene Expression Analysis of CML Patients across the Age Spectrum

Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells.CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO.At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p<0.0001), and 3.09-fold (p<0.0001), respectively, compared to pediatric CML CD34+ cells. Furthermore, DLC1, which is known as a negative regulator of Rho pathway, was significantly upregulated in pediatric CML CD34+ cells by 2.47-fold (p=0.0493) compared to adult CML CD34+ cells.These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. DisclosuresDavis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.

CML-271: Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors

Context: Cardiovascular (CVS) complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). Neutrophil extracellular traps (NETs) have previously been implicated in the pathogenesis of cancer-associated thrombosis. Objective: To assess NET formation in neutrophils from patients with CML, to characterize the role of reactive oxygen species (ROS) and peptidyl arginine deiminase 4 (PAD4) in NET formation in CML, and to assess the effects of TKIs on NET formation in CML. Design: Blood samples were obtained from patients with newly diagnosed CML and from age/gender-matched controls. NETs were assessed after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin (IO), with or without pre-treatment with imatinib, ponatinib, nilotinib, and dasatinib at clinically relevant concentrations. NET formation was assessed by morphology, immunofluorescence (IF), and the expression of proteins known to be associated with NET formation (NET-associated elastase, citrullinated histone H3 [H3cit], etc.). ER-HoxB8 conditionally immortalized murine myeloid progenitor cell line was differentiated into functional neutrophils and transduced with a BCR-ABL1 oncogene (or empty vector control). Results: Neutrophils isolated from treatment-naive patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with IO as assessed by IF and after stimulation with PMA as assessed by NET-associated elastase expression. Expression of H3cit, PAD4, and ROS was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation. Exposure to ponatinib significantly augmented NET-associated elastase and ROS levels as compared to control and other TKIs in neutrophils derived from patients with CML. BCR-ABL1 retrovirally transduced HoxB8-neutrophils demonstrated increased H3cit and myeloperoxidase expression, consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib and nilotinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. Conclusions: CML is associated with increased NET formation, which is further augmented by ponatinib and nilotinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the ER-HoxB8-BCR-ABL1 model, NET formation is PAD4- but not ROS-dependent.

Classification of chronic myeloid leukemia neutrophils by hyperspectral imaging using Euclidean and Mahalanobis distances

Chronic Myeloid Leukemia (CML) is a type of blood cancer which needs to be diagnosed in early stages to facilitate effective treatment. This necessitates quick, error free and automated diagnostic techniques. In this study, hyperspectral images have been analyzed using statistical distances to classify neutrophils from CML versus healthy blood samples. The statistical distances were used in multidimensional space offered by hyperspectral images. For computational efficiency, principal component analysis was used to achieve dimensionality reduction. The Euclidean distance method, and Mahalanobis distance method which compensates the variance of the target data distribution were used to classify CML neutrophils. The effectiveness of the proposed methods were tested and compared using experimental results. The Euclidean distance was found to be superior when it came to sensitivity in detecting CML neutrophils whereas the Mahalanobis distance was better at detecting healthy neutrophils and distinguishing CML neutrophils from healthy neutrophils.

Telomerase Inhibition Impairs Self-Renewal of ß-Catenin Activated Myeloproliferative Neoplasm Progenitors

IntroductionChronic phase myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML) and myelofibrosis (MF), arise from hematopoietic stem cells. However, they harbor varying propensities to undergo blast crisis (acute leukemic) transformation based on their capacity to give rise to tyrosine kinase inhibitor (TKI) resistant myeloid progenitors that activate self-renewal pathways, such as Wnt/b-catenin signaling. Because b-catenin has been reported to transcriptionally regulate human telomerase reverse transcriptase (hTERT), we investigated the capacity of a telomerase complex inhibitor, imetelstat, to prevent malignant progenitor self-renewal. The telomerase complex consists of hTERT, an RNA template subunit (TERC), and a protective shelterin scaffold. Imetelstat is a novel, first in class covalently lipidated 13-mer oligonucleotide telomerase inhibitor with clinical activity in myeloid malignancies. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms such as MF. However, the role of imetelstat in selectively eradicating self-renewing MPN progenitors has not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized MPN progenitor primagraft studies to investigate the ability of imetelstat to selectively inhibit malignant progenitor self-renewal at doses that spare normal progenitors as well as to determine the mechanism of action.Methods and ResultsWhen FACS-purified progenitors from human blast crisis (BC; n=9) were compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples, RNA-seq revealed upregulation of key genes in the TERT interactome suggesting a role for TERT activation in MPN progenitor blast crisis transformation. Human MPN progenitor supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 µM significantly inhibited (p <0.001, ANOVA) in vitro replating of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Also, imetelstat (5 µM) significantly reduced replating of MF (n=9) compared with normal progenitor (n=4) samples. Treatment of humanized mouse models, established with four different BC CML samples, with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in both bone marrow and spleen malignant progenitor burden compared with mismatch controls (p=0.003, and p=0.009, t test), and a significant reduction in human CD45+ cells in the splenic niche compared with mismatch controls (p=0.03, t test). In a MF model established in NSG-S mice, imetelstat also reduced human CD45+ cell engraftment (p=0.03, t test). Notably, imetelstat treatment spares normal human cells in humanized normal stem cell mouse models. Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat treated MF cells (p=0.18) and CML cells (p=0.4) compared with mismatch control. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human progenitors after imetelstat treatment (p=0.02) compared with vehicle control.ConclusionsNiche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated MPN progenitors to imetelstat in both in vitro and in vivo humanized pre-clinical MPN mouse models thereby providing a strong rationale for studies assessing eradication of malignant progenitors using imetelstat.Disclosures: This work was funded by the Moores Family Foundation, the Koman Family Foundation, the California Institute for Regenerative Medicine (DR1-01430), Janssen Research & Development, LLC, and by NIH NCI R21CA189705. DisclosuresHuang:Janssen Research & Development, LLC: Employment.

Mir-424 and Mir-503 Regulates Cobll1 Expression during the CML Progression

Up- or down-regulation of many miRNAs plays an important role in chronic myeloid leukemia (CML), however underlying mechanisms remain to be elucidated. Here, we report miRNA-424 and miR-503 regulates Cobll1 expression during CML progression.In previous our study, Cobll1 is a novel oncoprotein to associate with drug resistance and CML progression and highly expressed in blast crisis (BC). Also, overexpression of Cobll1 for blast formation and independently of BCR-ABL1 could be achieved in several ways. It could be by altering unknown signaling pathway(s), and being independent from BCR-ABL1. We searched for miRNAs that can regulate the expression level of Cobll1 to examine potential regulators of Cobll1 .To find candidate miRNAs of Cobll1, we were predicted to miRNA (miR-15a, miR-16, miR-103, miR-107, miR-424 and miR-503) by the algorithm in the TargetScan software. To validation target miRNA of Cobll1, we analyzed the expression levels of these miRNAs in CP and BC samples of 2 CML patients. Interestingly, miR-424 and miR-503 expression were significantly reduced in BC, which indicates that expression of Cobll1 and miR-424 or miR-503 is negative correlation.We next performed that eighty one CML samples (41 CP and 40 BC) were analyzed to determine the levels of the Cobll1 protein/miR-424 and miR-503 by Western blotting/qRT-PCR. Amazingly, we found that miR-424 and miR-503 were significantly downregulated highly expressed Cobll1 BC samples (n=21) compared to Cobll1 less expressed BC samples (n=19) and CP samples (n=41). Interestingly, there were down-regulations in expression of miR-424 and miR-503 that had high co-relation with Cobll1 expression in CML progression.To assess the effects of miR-424 or miR-503 regulates Cobll1 expression, we make ectopically expressing plasmids of wild-type Cobll1 3'-UTR and mutant Cobll1 3'-UTR. Transfection of miR-424 or miR-503 reduced luciferase activity in K562 cells ectopically expressing wild-type Cobll1 3'-UTR, but not a mutant Cobll1 3'-UTR. In addition, overexpression of miR-424 or miR-503 significantly reduced the endogenous Cobll1 levels in K562 cells. Consistently, the knockdown of Cobll1 by the miRNAs sensitized K562 cells to nilotinib-induced apoptosis. Taken together, our findings suggest that miR-424 and miR-503 plays a critical role CML by targeting directly targeting the Cobll1. These results provide the clue of miR-424 and miR-503 as a potential therapeutic target for CML progression by regulating the Cobll1 expression. DisclosuresNo relevant conflicts of interest to declare.

Longitudinal Detection of DNMT3AR882H Transcripts in Patients with Acute Myeloid Leukemia

Introduction The DNMT3A gene is recurrently mutated in normal karyotype acute myeloid leukemia (NK-AML) and the most common alteration (65% of cases) is located at residue R882 of methyltransferase domain. DNMT3A mutations often occur in association with other mutations such as FLT3 and NPM1 and while it is now generally accepted that AML patients with NPM1 or FLT3 -ITD mutations have favourable and poor outcome respectively, the impact of DNMT3A mutations on survival remains controversial. Recent studies reported that DNMT3A -mutated cells are detectable in patients with AML in long-lasting complete remission, questioning whether quantification of DNMT3A levels could be used to minimal residual disease (MRD). To address this issue, we carried out sequential monitoring studies of DNMT3AR882H levels in AML patients and compared our results with those obtained with multiparametric flow-cytometry (MPFC) and NPM1 molecular status and expression levels.Methods The mutational analysis of DNMT3AR882, NPM1 and FLT3 (ITD/TKD) was assessed at diagnosis in 556 patients with de novo AML (median age: 49 years, range 16-89 years) enrolled in clinical trials conducted by the Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) during the period 2012-2015. DNMT3AR882H molecular status was also studied in 100 patients with chronic myeloid leukemia (CML) and in 56 elderly healthy individuals. The expression levels of DNMT3AR882H and NPM1 Type_A mutation (NPM1mutA) were longitudinally analysed by qRT-PCR in 21 AML patients. For patients achieving complete morphologic remission (CR), DNMT3AR882Hlevels were compared with those of NPM1mutA and with MRD assessed by multiparametric flow cytometry (MPFC). W e then compared the levels of DNMT3AR882H and of MPFC-MRD in follow-up BM-samples from AML patients in CR , classified as MRDneg or MRDpos, according to threshold established based on previous work from our group (Buccisano et. al. Blood 2010). Finally, we evaluated the kinetics of DNMT3AR882H, NPM1mutA and FLT3 -ITD in 9 FLT3 -ITD+ve patients, using a FLT3 -ITD patient-specific qRT-PCR, and in 6 FLT3 -wt patients.Results Expression levels of DNMT3AR882H was also studied in sorted cell subpopulations from AML and CML patients and in healthy individuals. Distinct from NPM1mutA, DNMT3AR882H expression levels did not correlate with leukemic blast proportion assessed by MPFC (Figure 1) and decreased in AML samples only after induction treatment (Figure 2), while it was constantly detectable during remission and at relapse (Figure 3). Within FLT3 -ITD+ve AML, the kinetics of DNMT3AR882H paralleled that of NPM1mutA in 3 of 9 patients only. DNMT3AR882H expression levels were constant, independent from NPM1mutA in all 6 FLT3 -wt patients. Figure 4 shows 4 exemplary cases, with stable DNMT3AR882H levels during disease course in three of them. In CML patients, the DNMT3AR882H transcript was found only during follow-up in 4/100 patients, while it was undetectable at the time of CML diagnosis. DNMT3AR882H transcript levels were constantly low in all available CML samples. As previously reported, clonal hematopoiesis of indetermined potential (CHIP) was identified in 3/56 elderly healthy individuals. Similarly low expression levels of DNMT3AR882H were detected in blast and T-lymphocyte samples obtained from AML cases at CR and relapse phases, in CML patients and healthy individuals (Figure 5 and 6). There were no differences in DNMT3AR882H expression between MRDpos and MRDneg follow-up samples. DNMT3AR882H transcripts levels in MRDpos follow-up samples were significantly lower than those of samples harvested at diagnosis (Figure 6). These data show that DNMT3AR882H mutations probably arise in very early hematopoietic precursors in the context of CHIP. Interestingly, as reported in figure 6, DNMT3AR882H expression levels were similar in samples from CML patients, healthy individuals and AML follow-up samples.Conclusions In summary, the results of our study indicate that DNMT3AR882H quantification is unreliable as a tool for AML monitoring. In fact, together with its detection in CML and healthy subjects, the presence of DNMT3AR882H at constant levels detected in AML during active disease or CR clearly point to a lack of specificity of this abnormality for the leukemic AML blast population. [Display omitted] DisclosuresCicconi:TEVA: Speakers Bureau. Lo Coco:Lundbeck: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; TEVA: Honoraria, Speakers Bureau.

Expression and Functional Assessment of Some Featured Coding and Non-coding RNAs Encoded by 8q24 Chromosomal Region in CML Patients

Background: The association between the human chromosomal 8q24 region and cancer development remains dim. The proto-oncogene MYC is known as the most prominent target of this chromosomal region. However, numerous cancer-associated genetic alterations in the region extend beyond the MYC locus. Accordingly, it is likely that the MYC oncogene is not the only target of these carcinogenesis-related alterations. Objectives: In the present study, the expression of MYC and the correlation between MYC and two non-coding RNAs, namely PVT1 (circular and linear forms) and CASC11, which are residents of the 8q24 region in the MYC neighborhood, were investigated in chronic myeloid leukemia (CML). Methods: Real-time polymerase chain reaction (PCR) was used to assess BCR-ABL transcripts and categorize positive and negative (normal) samples for CML. Afterward, real-time PCR was exploited to evaluate the expression of different genes, including MYC, linear PVT1, circular PVT (CircPVT1), CASC11, and ACTB in CML and normal samples. Results: We found that the expression of linear PVT1 is significantly increased in CML compared with normal samples. However, CircPVT1, CASC11, and MYC did not show significantly altered expression between CML and normal groups. The experimental and in silico analyses of the correlation coefficients of gene expressions suggested changes in the correlations between the gene expressions in CML compared with normal samples. We also assessed the miR-trapping potential of PVT1 and CASC11 and the possible effects of these interactions on signaling pathways. Our findings indicated that these lncRNAs could have a possible regulatory link with critical pathways associated with leukemogenesis. Conclusions: Our results indicate that non-coding genes surrounding MYC within the 8q24 region might have regulatory roles in CML carcinogenesis.

Simvastatin enhances the efficacy of nilotinib in chronic myeloid leukaemia by post-translational modification and drug transporter modulation

The resistance of chronic myeloid leukaemia (CML) to tyrosine kinase inhibitors (TKIs) remains a significant clinical problem. Targeting alternative pathways, such as protein prenylation, is known to be effective in overcoming resistance. Simvastatin inhibits 3-hydroxy-3-methylglutaryl-CoA reductase (a key enzyme in isoprenoid-regulation), thereby inhibiting prenylation. We demonstrate that simvastatin alone effectively inhibits proliferation in a panel of TKI-resistant CML cell lines, regardless of mechanism of resistance. We further show that the combination of nilotinib and simvastatin synergistically kills CML cells via an increase in apoptosis and decrease in prosurvival proteins and cellular proliferation. Mechanistically, simvastatin inhibits protein prenylation as shown by increased levels of unprenylated Ras and rescue experiments with mevalonate resulted in abrogation of synergism. The combination also leads to an increase in the intracellular uptake and retention of radio-labelled nilotinib, which further enhances the inhibition of Bcr-Abl kinase activity. In primary CML samples, this combination inhibits clonogenicity in both imatinib-naive and resistant cells. Such combinatorial effects provide the basis for utilising these Food and Drug Administration-approved drugs as a potential clinical approach in overcoming resistance and improving CML treatment.