BCR-ABL Gene Research Articles

Abstract 4184: Drug set enrichment analysis : A computational approach to identify functional drug sets

Abstract Most drug-testing approaches published so far focus on identifying a single drug that shows favorable response and is associated with a known cancer biomarker such as the drug Imatinib in BCR-ABL gene fusion positive cells. We developed and applied drug set enrichment analysis (DSEA) to find enriched patterns or statistically significant similarities (overlaps) between the drug responses of a test sample against a cohort of 182 previously screened cancer samples. The samples studied included established (ATCC) cancer cell lines, drug-resistant cancer cell models, ex-vivo patient cancer cells in primary cultures, including conditionally reprogrammed cancer cells from patients. DSEA is adopting Gene Set Enrichment Analysis statistics commonly used for gene expression analysis to high throughput drug testing data. Our drug screening (Pemovska et al., Cancer Discovery, 2013) was done with a panel of 306 established (FDA-approved) and emerging targeted cancer drugs such as tyrosine-kinase inhibitors (e.g. EGFR, PDGFR, BRAF, MET), S/T-type inhibitors, (e.g. MEK, Plk1, Akt, Aurora, Chk1), and inhibitors of other pathways (HDACs, Hh, BCL2, PI3K, PARP) and many others. The readout was based on viability of cells after a 72 hour culture. The DSEA approach is based on taking the top most sensitive drugs (above a defined sensitivity score cut-off) in an individual cancer sample and then identifying overlapping drug response profiles in previously screened reference samples. Our hypothesis is that the most sensitive drug sets in any given sample tend to show similar response profiles in a cohort of similar samples. We convey the correlations and drug set enrichment analysis results as dendrogram trees, plots and tables with enrichment and significance scores. Interestingly, our results show that clustering of drug sensitivity testing data does not place all cancer cell line samples within well-established subgroups based on biological features or histological origin. We find a similar tendency in ex vivo patient samples. Therefore, comprehensive drug response profiles seen may reveal novel biological data that reflect pharmacologically-relevant, phenotypic cancer cell states. DSEA could also provide novel means to subtype previously poorly characterized cancer samples based on their drug response profiles and thereby in the future facilitate the choice of therapies to patients whose cancers repond in an atypical way as compared to the expectations based on anatomical origin or genomic composition. Citation Format: John Patrick Mpindi, Dimitry Bychkov, Yadav Bhagwan, Disha Malani, Hirasawa Akira, Khalid Saeed, Susanne Hultsch, Sara Kangaspeska, Astrid Murumägi, Caroline A Heckman, Kimmo Porkka, Tero Aittokallio, Krister Wennerberg, Päivi Östling, Olli Kallioniemi. Drug set enrichment analysis : A computational approach to identify functional drug sets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4184. doi:10.1158/1538-7445.AM2014-4184

Safranal, a Crocus sativus L constituent suppresses the growth of K-562 cells of chronic myelogenous leukemia. In silico and in vitro study

Crocin, a main constituent of Crocus sativus L (saffron), has been found to inhibit the growth of K-562 human chronic myelogenous leukemia (CML) cells expressing Bcr-Abl protein tyrosine kinase activity. The aim of our study is to investigate the ability of the bioactive saffron's constituents, crocin (CRC) and safranal (SFR), to inhibit the Bcr-Abl protein activity employing an in silico approach, as well as the in vitro effect of these compounds on K-562 growth and gene expression of Bcr-Abl. In silico molecular docking studies revealed that mostly SFR can be attached to Bcr-Abl protein, positioned inside the protein's binding cavity at the same place with the drug used in the treatment of CML, imatinib mesylate (IM). The predicted polar interactions and hydrophobic contacts constructing a hydrophobic cavity inside the active site, explain the observed inhibitory activity. Cytotoxicity experiments showed that SFR and CRC mediate cytotoxic response to K562 cells. In vitro studies on the expression of Bcr-Abl gene revealed that SFR and in a lesser degree IM inhibited the expression of the gene, while in contrast CRC induced an increase. The ultimate goal was to evaluate the existence of a potential antitumor activity of saffron's constituents SFR and CRC.

Inhibition of 32Dp210 cells harboring T315I mutation by a novel derivative of emodin correlates with down-regulation of BCR-ABL and its downstream signaling pathways.

The clinical outcome of chronic myeloid leukemia (CML) patients has been changed dramatically due to the development of imatinib (IM). However, the emergence of IM resistance, commonly associated with point mutations within the BCR-ABL kinase domain, remains a major clinical problem. Here, we investigated the effects of E35, a novel derivative of emodin, on the IM-resistant 32Dp210-T315I cells. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide and colony formation assay. Induction of apoptosis was confirmed by DNA fragmentation assay and annexin V/PI staining assay. Real-time quantitative PCR was used to access the BCR-ABL gene expression. Changes of related signaling molecules were detected through Western blot. E35 was found to potently inhibit proliferation of 32Dp210-T315I cells with an average IC50 of 2.4 µM at 48 h. Colony formation was almost fully suppressed in 1.0 μM E35 group. DNA fragmentation and annexin V/PI staining assay exhibited the typical DNA fragmentation and the increased proportion of early apoptotic cells, respectively. The induction of apoptosis was associated with increase of Bax to Bcl-2 expression ratio and activation of caspase cascades involving decrease of pro-caspase 9 and pro-caspase 3 and increase of PARP cleavage. The protein expression of P210(BCR-ABL) and p-P210(BCR-ABL) was down-regulated in the presence of E35, although the mRNA levels remained almost unchanged. Moreover, the activation of the P210(BCR-ABL) downstream signaling pathways including CrkL, Akt/mTOR and MEK/ERK was fully suppressed by E35. Our study indicated that E35 might be a potential antileukemia agent against IM resistance in CML.

Anti-rheumatic agent auranofin induced apoptosis in chronic myeloid leukemia cells resistant to imatinib through both Bcr/Abl-dependent and -independent mechanisms.

Resistance to Imatinib mesylate (IM) is an emerging problem for patients with chronic myelogenous leukemia (CML). T315I mutation in the Bcr-Abl is the predominant mechanism of the acquired resistance to IM and second generation tyrosine kinase inhibitors (TKI). Therefore it is urgent to search for new measures to overcome TKI-resistance. Auranofin (AF), clinically used to treat rheumatic arthritis, was recently approved by US Food and Drug Administration for Phase II clinical trial to treat cancer. In contrast to the reports that AF induces apoptosis by increasing intracellular reactive oxygen species (ROS) levels via inhibiting thioredoxin reductase, our recent study revealed that AF-induced apoptosis depends on inhibition of proteasomal deubiquitinases (UCHL5 and USP14). Here we report that (i) AF induces apoptosis in both Bcr-Abl wild-type cells and Bcr-Abl-T315I mutation cells and inhibits the growth of IM-resistant Bcr-Abl-T315I xenografts in vivo; (ii) AF inhibits Bcr-Abl through both downregulation of Bcr-Abl gene expression and Bcr-Abl cleavage mediated by proteasome inhibition-induced caspase activation; (iii) proteasome inhibition but not ROS is required for AF-induced caspase activation and apoptosis. These findings support that AF overcomes IM resistance through both Bcr/Abl-dependent and -independent mechanisms, providing great clinical significance for cancer treatment.

Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR-ABL1 fusion gene.

For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell(HPC) disorder. However, it remains to be determined whether BCR-ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR-ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR-ABL1 fusion gene in FACS-sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97%615%), basophils (95%612%), eosinophils (90%68%), CD341 precursors cells (90%67%),monocytes (84%630%), nucleated red blood cells (87%624%), and mast cells (77%633%). By contrast, MPC(30%634%), B-cells (15%627%), T-lymphocytes (50%626%), and NK-cells (35%634%) were involved at lower percentages. In 8/18 CML patients, 2 tumor BCR-ABL11 subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD341 cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR-ABL1 fusion gene inMPC, CD341 precursors, and other different BM hematopoietic myeloid cell lineages from CML patients,including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes.Interestingly, involvement of MPC was restricted to the ancestral BCR-ABL11 subclone.

Successful treatment of Philadelphia chromosome-positive mixed phenotype acute leukemia by appropriate alternation of second-generation tyrosine kinase inhibitors according to BCR-ABL1 mutation status

Philadelphia chromosome-positive mixed phenotype acute leukemia (Ph(+)MPAL) is a rare type of acute leukemia having myeloid and lymphoid features. In the present study, we describe the successful treatment of a 71-year-old Japanese female patient with Ph(+)MPAL by the alternation of second-generation tyrosine kinase inhibitors according to BCR-ABL1 mutations. The patient survived in her third complete remission (CR) for over 4 years. In her first CR, the patient was treated with multiple-agent chemotherapy and underwent maintenance therapy with imatinib and monthly vincristine and prednisolone (VP). At the first relapse, an examination of the bone marrow revealed a transformation into acute lymphoblastic leukemia and an F317L mutation in BCR-ABL1 gene, which responded preferentially to nilotinib over dasatinib. She achieved second CR, and nilotinib with VP therapy was selected for maintenance treatment. At second relapse, BCR-ABL1 mutational analysis revealed Y253H mutation instead of F317L mutation, resulting in resistance to nilotinib. The patient achieved third CR with dasatinib and VP therapy, and maintained CR with this treatment. This suggests that appropriate alternation of TKIs may contribute to long-term survival in elderly patients with Ph(+)MPAL.

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro

To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro. 32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells. C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC₅₀ at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC₅₀ at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells. C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

Constitutive 53BP1/γH2AX foci are increased in cells of ALL patients dependent on BCR-ABL and TEL-AML1 preleukemic gene fusions.

Childhood leukemia arises from hematopoietic stem cells by induction of mutations. Quite often chromosomal translocations arise prenatally as first key event in multistage process of leukemogenesis. These translocations result in so called preleukemic gene fusions (PGFs), such as BCR-ABL and TEL-AML1, which generate hybrid proteins with altered properties. Critical DNA damage resulting in translocations are DNA double-strand breaks (DSBs). BCR-ABL and TEL-AML1 were shown to be associated with increased constitutive DSBs in various model systems. We analyzed cells from peripheral blood and CD34-/CD34+ cells from bone marrow of pediatric acute lymphoblastic leukemia (ALL) patients harboring BCR-ABL or TEL-AML1. We used sensitive technique that is based on automated enumeration of DSB co-localizing proteins γH2AX and 53BP1, which form so called DNA repair foci. We found that level of constitutive γH2AX/53BP1 foci is significantly higher in cells of ALL pediatric patients than in healthy subjects. There was also significant increased level of constitutive γH2AX/53BP1 foci in cells from ALL patients harboring BCR-ABL or TEL-AML1 compared to patients without PGFs. The same increase was observed regardless cell type for both PGFs. Our data on increased DSB levels in the BCR-ABL/TEL-AML1 patient's cells support a model where BCR-ABL/TEL-AML1 induces DNA instability through facilitating mutagenesis and appearance of additional genetic alterations driving leukemogenesis.

Study on the treatment of the p15 gene combined with Bcr-abl-specific siRNA and STI571 for chronic myeloid leukemia.

The aim of this study was to investigate the effect of the p15 gene combined with Bcr-abl-specific siRNA and STI571 on the proliferation, cell cycle and apoptosis of K562 chronic myeloid leukemia cells. Using the gene sequence results, we amplified the p15 gene from normal peripheral blood by RT-PCR, and constructed a p15-pcDNA3.1 vector. The K562 cell line with G418 resistance was screened, synthesized and transfected for bcr-abl gene fusion point for 21-nt siRNA. In p15-pcDNA3.1-K562 cells, the growth rate was slower than that of the control K562 cells, G0/G1-phase was increased and S-phase was decreased significantly. In the siRNA group, bcr-abl fusion gene expression was significantly decreased in K562 cells accompanied by the downregulation of BCL-xL protein expression and G1-phase arrest. Cell survival rate was significantly decreased compared with the sole p15-K562 cell group and the sole RNA interference-K562 cell group. In the combination of p15-pcDNA3.1-K562 cells with STI571, the proportion of apoptosis was significantly increased and the cell survival rate was significantly decreased compared with the p15-K562 cell group and STI571-K562 cell group. siRNA at 30 pM combined with 0.5 μM STI571 promoted apoptosis compared with sole application. The p15 gene combined with siRNA had a synergistic effect on the inhibition of proliferation and the induction of apoptosis in K562 cells. Exogenous p15 protein expression combined with STI571 appeared to have a synergistic effect on proliferation inhibition and apoptosis induction in K562 cells. The combination of low-dose RNA interference with STI571 showed a synergistic effect in inducing apoptosis.

Imatinib resistance in chronic myelogenous leukemia: an emerging challenge

Resistance to imatinib is a significant clinical issue, and the underlying mechanism of this resistance is multifactorial. The efficacy of imatinib in chronic myeloid leukemia (CML) in achieving a high remission rate and improving prognosis has seriously been challenged by the development of mutants of BCR-ABL gene, which resist the action of imatinib, which is a tyrosine kinase inhibitor. We present here a case of a 35-year-old male, a known case of CML on imatinib therapy, the patient eventually landed in blast crisis and succumbed to the disease and secondary infections.

Status of Leptin in MBCR-ABL p210 Positive Chronic Myeloid Leukemia Patients Before and After Imatinib Therapy: A Conflicting Scenario

Chronic myelogenous leukemia (CML), a myeoloproliferative disorder, is characterized by the presence of the fusion gene BCR-ABL in hematopoietic cells. Leptin, considered a link between cancer and obesity, has been reported to be actively involved in hemopoiesis and pathophysiology of CML. There are few and conflicting reports about the status of serum leptin levels and recently alteration in leptin has been reported due to imatinib mesylate. Leptin and CRP were estimated in 30 (male: 20; female: 10) newly diagnosed and confirmed MBCR- ABL p210 positive CML patients before and after 3 months of therapy by commercial enzyme linked immunosorbent assays. Leptin levels were compared with 30 (male: 20; female: 10) age matched healthy controls accounting for the differences due BMI and gender. Leptin/BMI ratio was significantly raised in both male and female chronic phase patients as compared to controls (p < 0.001, p = 0.048) and accelerated phase patients as compared to controls (males, p < 0.001; females, p < 0.001). The normal gender difference and dependence on BMI was lost in patients. In patients, who failed to achieve hematological baseline, leptin/BMI was higher only in male patients (p = 0.012). Leptin/BMI also correlat- ed with TLC and blast percentage (TLC, R2 = 0.412, p = 0.001; Blast %, R2 = 0.408, p < 0.001). There was no correlation between leptin and CRP levels. Levels decreased significantly after complete hematological remission in both males and females (p = 0.001, p = 0.028). Levels after 3 months of imatinib therapy were significantly higher than controls in all patients not in remission (males, p < 0.001; females, p = 0.018) but only in male patients in re- mission (p = 0.002). Leptin levels were increased in CML patients. The findings suggest a possible role of leptin in patho- genesis of CML or disease progression independent of inflammatory state or reactionary rise. Imatinib itself may increase leptin levels, and, as leptin plays an active role in the pathophysiology of CML, this conflicting scenario needs further investigation. Alterations in leptin need to be investigated cautiously accounting for confounding and differences due to BMI and gender.

What Does a Deep Molecular Response Signify?

What Does a Deep Molecular Response Signify?

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Identification Of Mutations In Bcr-Abl Gene In Newly Diagnosed Indian Patients With Chronic Myelogenous Leukemia (CML)

Background & ObjectivesMutations in Bcr-Abl gene is responsible for resistance after Imatinib treatment in 30-60% of the cases. Currently the data does not support testing for these mutations in Imatinib naïve cases. However there is a data emerging that suggest that base line mutations in the Bcr-Abl gene do exist even before Imatinib is started, especially in patients with high SOKAL score. In India the failure rate of Imatinib is higher in comparison to western literature because patients are detected for CML chronic phase (CP) at a later stage. This study was designed to identify mutations in Abl-KD in Indian CML patients with no previous exposure to tyrosine kinase inhibitor. MethodsThis study targeted a cohort of CML-CP patients (n=47) without prior TKI exposure. KD mutations were analyzed by direct sequencing. ResultsA total of 47 samples were evaluated, out of which 2 samples were identified to have the same variation. This heterozygous missense variation (A206D) in the Abl1 gene was located on SH2 domain of Exon 4. The change was from C>A in codon 206, which caused a protein change from Alanine to Aspartic acid (p.A206D). By using the mutation taster software, this change was predicted to be disease causing with a high probability of .99 (∞1). One of the patients died within one year of diagnosis in spite of imatinib treatment. Interpretation & conclusionA novel mutation with putative pathogenicity was identified in the TKI-naïve Indian CML patients, supporting the concept that naturally occurring KD mutations are present in leukemic cell prior to drug exposure. Disclosures:No relevant conflicts of interest to declare.

Defining The morphological Appearance Of ETV6-Abl Diseases. Evaluation Of a Case

A 30 year old female presented@30 weeks pregnancy with a few circulating myeloid blasts in her blood but a normal full blood count. She had previously used an epipen for acute allergic reactions and had bone pain at presentation. There was splenomegaly on examination. Bone marrow aspirate was extremely difficult to obtain but trephine roll showed eosinophils, basophils, occasional mast cells and occasional blasts A population of 23% hypergranulated basophilic cells were seen on aspirate whose aetiology is not clear and are assigned to the basophil/mast cell series. For want of a better term these cells are called Finella's cells. Histological examination of trephine biopsy showed extreme hypercellularity, Grade III/IV reticulin fibrosis large numbers of eosinophils and basophils with mast cells confirmed by CD 117 staining. The large hypergranulated basophilic cells (Finella's cells) were not visible on trephine staining. An isodicentric X on cytogenetic analysis and a bcr-abl probe showed a split abl with 2 signals. An ETV6-ABL1 gene rearrangement was present. The c-kit D816V mutation was not identified nor was FIP1L1-PDGFRA present. Subsequently the morphologic features described above allowed the suspicion in a further case.Various appearances of the bone marrow and blood allow the suspicion of a particular disease which then leads to the performance of the diagnostic test to confirm the suspected genetic abnormality. This is epitomised in the diagnosis of chronic myeloid leukaemia based on a high white cell count, myelocyte peak and excess of myeloid cells with an increase in cells such as eosinophils and basophils. The diagnosis is then made by demonstrating the presence of the Philadelphia chromosome or bcr-abl gene rearrangement.In contrast in cases with an ETV6-abl, the patient has a white cell count that is not markedly increased with a leucoerythroblastic blood film and a bone marrow examination that shows a superficial resemblance to a chronic myeloproliferative disease with marked myelofibrosis and large numbers of eosinophils and basophils some of which are morphologically abnormal. Megakaryocytes when seen show discrete nuclear fragments and hypolobation and eosinophils and basophils are increased leading to the case being mistakenly diagnosed as an eosinophilic disorder rather than a specific disease entity i.e ETV6-abl. It cannot be classified with the mastocytic leukaemia's as the number of masts cells is not greater than 5% and they are not clustered but loosely distributed. The aetiology of Finella's cells are unclear. Mast cells in blood smears are easily identifiable by their large granules which disfigure the nuclear outline and a central area of pallor or by their “slipper” shape and granule content. In this case the cells are larger than mast cells circular, have no central area of pallor,the excessive granularity masking the nuclear outline and leading one to question whether a PML-RARA fusion is present which it is not. The presence of eosinophils, basophils and these cells should make the viewer suspect an ETV6-abl disorder which is normally a cryptic fusion which will not be identified without use of the appropriate FISH/ RT-PCR test. The disease appears to undergo blast transformation as seen with CML and early transplantation is recommended as happened successfully with our case.Although several other cases have been reported in the literature by various authors and called bcr-abl negative CML, myeloproliferative neoplasms, myeloproliferative disorders with eosinophilia etc peripheral blood eosinophilia is not present and FIP1L1-PDGFRA is not present. The morphologic features that lead one to suspect the presence of theETV6-abl have not been previously delineated. THE ETV6-ABL fusion protein has tyrosine kinase activity and responds to imatinib or second generation TKI's and therefore demonstrating its presence when faced by the classic morphologic appearance is of clinical importance. Disclosures:No relevant conflicts of interest to declare.

The Role Of Splenectomy In The Treatment Of Myelofibrosis

BackgroundSplenectomy (SE) at the Myelofibrosis (MF) is one of the difficult operations on the techniques and perioperative complications. There are a lot of complications such as inflammatory and adhesive processes, a massive bleeding associated with disorders of hemostasis in thrombocytopenia or thrombocytosis, enlargement of varicose veins, concomitant pathology. A SE doesn't cure myelofibrosis but may be performed in some cases. Success is unpredictable, and the operative mortality of SE is on the order of 10%. ObjectivesEvaluate the clinical and hematologic SE efficiency in patients with MF, resistant to traditional treatment. Patients and methodsWe analyzed the medical case history of 52 patients with MF, which were observed in the National Research Center for Hematology from 2004 to 2013. All patients were performed SE. 47 patients diagnosed with primary MF, and 4 – post-polycitaemic MF and 1 patient post-trombocitaemic MF. PMF was diagnosed according to the criteria established by WHO (2008), which include bone marrow fibrosis, and splenomegaly leyko-eritroblastosis in peripheral blood. The average age at diagnosis was 47 years (24 – 78 years), and before the SE – 53 (25 – 79 years). Women and men ratio was 1.3:1. According to the predictive model of the Dynamic International Prognostic Score System - (DIPSS) 38 (73%) patients before the SE attributed to intermediate risk 2, 14 (27%) patients – high risk. BCR-ABL gene not found a single patient, and the gene mutation JAK2V617F at different stages of the disease is detected in 53% of cases. Genetic adverse factors are not taken into account because of the small number of studies. Massive and huge splenomegaly was present in 37 (71%) patients. Spleen weight 0.9 to 2.9 kg is removed in 15 (29%) patients, from 3 to 7 kg is removed in 35 (67%) patients. In two cases, removal of the spleen weight of 10 and 11 kg. Statistical analysis were done using SAS 9.3 and JMP 10.0 (SAS Institute Inc., Cary, NC). ResultsDisease duration was about 76 months and ranged from 1 to 240 months. Surgical complications occurred in 21 patients, including bleeding 3, thrombosis 1, infection 2. Surgical complications from SE in intra-and early postoperative period was 8% from 1978 to 2003. Surgical complications from SE in intra-and early postoperative period was not fixed from 2004 to 2013. After SE complications were hepatomegaly, resistant thrombocytosis (platelet count ≥ 600 × 109/L), leukocytosis (white blood cell count greater than 10 x 109/l) and blast transformation of the disease. More than 80% of patients after SE had no symptoms of intoxication. 22 patients with MF had transfusion dependent anemia, 11 (50%) had disappeared transfusion requirements after SE in and the effect lasted for about a year. Only 4 of the 15 patients with thrombocytopenia who underwent SE due to refractory thrombocytopenia were observed a significant increase in platelet count above 100h109 / L, for an average of 6 months. The possibility of the resumption of long-term cytoreductive therapy appeared in 30 (58%) patients. Currently alive 19 (37%) patients, mean follow-up was 37 months (4 to 72 months) after the SE and of 105 (12-264) months after diagnosis. 33 (63%) patients died during the observation period after SE (1 – 84 months, mean – 27 months). Among these, 27 (82%) patients died of transformation disease and 6 (18%) died of comorbidity. 19 (37%) patients, with a mean follow-up of 37 months after SE (from 4 to 72 months) continue treatment with hydroxyurea. Median survival after SE is 3 years (Figure 1), with a median overall survival - 11 years (Figure 2). [Display omitted] [Display omitted] ConclusionDespite the potential complications SE is an effective palliative method for patients with MF in the absence of leukemic progression. SE indicated in patients with drug refractory MF, acute constitutional symptoms, anemia, transfusion-dependent, and portal hypertension. Modern methods of surgery, as well as supporting therapy in the perioperative period can safely perform the SE. Disclosures:No relevant conflicts of interest to declare.

Synergistic Cytotoxicity Between Panobinostat and Ponatinib In Imatinib-Resistant Chronic Myeloid Leukemia Cells

Current first-line treatment options for chronic myeloid leukemia (CML) include imatinib (IM) and the second-generation agents nilotinib and dasatinib. Despite the effectiveness of these tyrosine kinase inhibitors, a small percentage of chronic-phase CML patients are primarily refractory or acquire secondary resistance to these agents. Moreover, the prognosis of patients in blast crisis is still poor despite the use of the current treatment modalities because of drug resistance. The major mechanism of drug resistance is the re-activation of ABL kinase either through mutations in the BCR-ABL gene or by BCR-ABLgene amplification.A novel pan-histone deacetylase inhibitor, panobinostat (formerly LBH589), induces the acetylation of heat shock protein 90, thereby inhibiting its chaperone function in association with its client proteins BCR-ABL, leading to the degradation of BCR-ABL. A new pan–ABL tyrosine kinase inhibitor, ponatinib, is a promising therapeutic option in patients with all kinds of BCR-ABLmutation including T315I. It was thus hypothesized that the combination of panobinostat and ponatinib exerts synergistic cytotoxicity against IM-resistant cells through a mechanism of action different from that of each agent. To test this hypothesis, K562/IM-R1 and Ba/F3/T315I cell lines were evaluated for the cytotoxicity of panobinostat and ponatinib in vitro. K562/IM-R1 cells, established in our previous study, showed BCR-ABL overexpression due to BCR-ABL gene amplification. The Ba/F3/T315I cell line showed BCR-ABL with a T315I mutation.The XTT proliferation assay revealed that K562/IM-R1 cells were 12-fold more IM-resistant (50%-inhibitory concentration (IC50), 7.6 µM) than K562 cells (IC50, 0.6 µM). Ba/F3/T315I cells were refractory to IM treatment (IC50, 34.1 µM) compared with Ba/F3 cells (IC50, 3.4 µM). Panobinostat overcame IM-resistance and inhibited similarly the growth of K562, K562/IM-R1, Ba/F3, and Ba/F3/T315I cells, with IC50 values of around 50 nM (40.0 - 51.0 nM). Ponatinib inhibited the growth of both K562/IM-R1 cells (IC50, 4 nM) and Ba/F3/T315I cells (25 nM) as potently as parental K562 cells (IC50, 2 nM) and Ba/F3 cells (IC50, 5 nM). Importantly, the combination of panobinostat and ponatinib exhibited enhanced growth inhibition effects on all cell lines. The IC50 values for this combination were 0.7 nM for K562 cells, 1.3 nM for K562/IM-R1 cells, 3.7 nM for Ba/F3 cells, and 10 nM for Ba/F3/T315I cells. The combination index clearly showed synergism, with values of 0.5 for K562 cells, 0.28 for K562/IM-R1 cells, 0.9 for Ba/F3 cells, and 0.4 for Ba/F3/T315I cells. When the cells were treated with 10 nM panobinostat or 10 nM ponatinib for 48 h alone or in combination, the combination of the 2 agents led to greater-than-additive apoptotic cell death than each agent alone in all cell lines, evaluated by annexin V-positivity.Western blotting was used to evaluate the protein expression levels of BCR-ABL and phospho-BCR-ABL in cells after treatment with panobinostat or ponatinib or both in combination. BCR-ABL expression was greater in K562/IM-R1 than K562 cells. Phospho-BCR-ABL expression was not inhibited by IM in K562/IM-R1 or Ba/F3/T315I cells. However, ponatinib inhibited the autophosphorylation of BCR-ABL in these cell lines. Treatment with panobinostat reduced the BCR-ABL and phospho-BCR-ABL expression levels in K562/IM-R1 and Ba/F3/T315I cells. The combination of the 2 agents augmented inhibition of the autophosphorylation of BCR-ABL in these IM-resistant cell lines.The activity of histone deacetylase, determined using the HDAC assay Kit (Active Motif, Carlsbad, CA, USA), was inhibited by panobinostat in all cell lines regardless of IM sensitivity. In comparison, IM did not alter cellular histone deacetylase activity. Upon treatment of K562 cells with panobinostat, the protein expression levels of acetylated histone H3 and H4 were increased, suggesting the consequence of the inhibition of histone deacetylase.In conclusion, we firstly reported that panobinostat and ponatinib demonstrated synergistic cytotoxicity against IM-resistant cell lines not only due to BCR-ABL gene amplification but also BCR-ABL T315I mutation. The synergism is attributable to the greater inhibition of ABL kinase activity through a mechanism of action different from that of each agent. Disclosures:No relevant conflicts of interest to declare.

A Pilot Study Of Bone Marrow and Adipose Tissue – Derived Mesenchymal Stem Cells In Chronic Myeloid Leukemia (CML); Mesenchymal Stem Cells Of The Bone Marrow Harbor The Bcr-Abl Fusion Gene In a CML Patient

BackgroundChronic myeloid leukemia (CML) is a disease of the haemopoietic stem cell, hence its hallmark chromosomal rearrangement is detected in different haemopoietic lineages. Due to reported altered adhesion of haemopoietic cells to bone marrow stroma in CML, the cytogenetic profile and properties of bone marrow and adipose tissue mesenchymal cells have been the interest of our current study. Previous research has shown that the BCR-ABL fusion gene is not present in bone marrow mesenchymal stem cells; however, it has been detected in endothelial cells and in Flk1+ hemangioblastic progenitors. AimsTo study the properties of mesenchymal stem cells in CML, we initially investigated the presence of the BCR-ABL fusion gene in bone marrow and adipose tissue mesenchymal stem cells (MSCs) of patients with CML at diagnosis. Although previous studies have yielded negative results, the numbers of patients studied have been limited. Moreover, the presence of BCR-ABL in hemangioblastic progenitors in some cases of CML patients highlights the possibility that the lesion can sometimes be detected in a progenitor cell much more primitive than the haemopoietic stem cell. Adipose tissue MSCs were studied concomitantly with those of bone marrow in order to establish possible cell motility between the two compartments during disease evolution. MethodsWe isolated mesenchymal stem cells from bone marrow and adipose tissue mononuclear cells of four CML patients via cell culture. MSCs were isolated from the mononuclear cell fraction via serial medium changes removing non-adherent cells, followed by at least three passages. Patient samples were collected before the onset of therapy; three of the patients were diagnosed in chronic phase and one in blast crisis. High purity of MSCs from haemopoietic cell contaminants (>94% in all cases) was confirmed by flow cytometry; mesenchymal stem cells were positive for CD90, CD73 and CD105 and negative for CD34 and CD45. FISH analysis was performed to detect the BCR-ABL gene (probe LSI BCR/ABL1, ES Dual Color Translocation, Vysis). For each sample, 500 nuclei were examined. ResultsThe BCR-ABL fusion gene was detected in 15% of the bone marrow MSCs' nuclei of one patient, who had chronic phase CML. The CD34+/CD45+ cell population percentage in the final MSC population isolated from this patient was only 1,5% on flow cytometry analysis, which means that this result cannot be accounted for by haemopoietic cell contamination of MSCs. The other three patients did not carry the translocation in their bone marrow MSCs upon FISH analysis. Adipose tissue MSCs of all four patients were negative for BCR-ABL. ConclusionsWe have shown the presence of the BCR-ABL fusion gene in bone marrow mesenchymal stem cells of one CML patient. This result implies an occurrence of the t (9;22) (q11;134) translocation at a more primitive level in some cases of the disease, so that the bone marrow stroma can be involved in the malignant clone. Because there is still limited knowledge about the role of MSCs, whether BCR-ABL–positive or negative, in the development of the malignant haemopoietic clone in CML, our study will be extended to study the cytogenetic and gene expression profile of mesenchymal stem cells in a larger number of CML patients. Disclosures:No relevant conflicts of interest to declare.

Role Of Insulin Like Growth Factor mRNA Binding Protein-3 (IGF2BP3) In Mixed Lineage Leukemia (MLL) Positive B-Cell Lymphomas

Oncogenic transformation of early B-cell progenitors leads to the human disease of B-acute lymphoblastic leukemia or B-ALL, which affects both children and adults. Among the different subtypes of B-ALL, defined by particular cytogenetic anomalies, there are two which are difficult to treat and have a dismal prognosis. These are B-ALL with chromosomal translocation t (9; 22)/BCR-ABL and MLL gene rearrangements, which show distinctive gene expression profiles.Gene expression is now known to be significantly regulated by post-transcriptional mechanisms. These involve RNA binding proteins and microRNAs. Deregulation of microRNAs as well as RNA binding protein expression is associated with numerous cancers. Here, we hypothesized that RNA binding proteins may be important in regulating gene expression in MLL rearranged leukemias.To examine this hypothesis, we undertook a microarray study examining the expression of both protein-coding and non-coding genes in B-ALL, including MLL translocations. A total of 44 samples were used for the microarray. Supervised class prediction was carried out using the R library of prediction analysis for microarrays (PAM). One of the most significantly differentially expressed genes was Insulin Like Growth Factor mRNA Binding Protein-3 (IGF2BP3). The expression of IGF2BP3 was highest in the MLL rearranged B-ALL group. IGF2BP3 is an oncofetal protein known to be highly expressed in a number of epithelial malignancies such as glioblastomas. IGF2BP3 has been known to bind to the 5'-UTR and stabilize mRNAs like CD44 the expression of which correlates with epithelial tumors metastasis. IGF2BP3 has been shown to bind to the Insulin like Growth Factor-2 (IGF-2) mRNA and enhance translation in glioblastomas.We confirmed the expression of IGF2BP3 and CD44 in these 44 tumor samples and 90 other B-cell lymphoma samples by RT-qPCR. This corroborated with our previous data showing that the expression of both these genes is significantly higher in the group with MLL translocations. In the MLL rearranged leukemias, there was a significant correlation between the expression of CD44 and IGF2BP3. Interestingly however, there was no significant difference in the expression of IGF2 mRNA between these different subsets, indicating either that IGF2BP3 might be acting on IGF-2 mRNA at the translational level or that IGF-2 regulation may be cell-type specific.To evaluate whether IGF2BP3 affects the growth of B-ALL cells, we used NALM6, a B-ALL cell line which expresses IGF2BP3. We generated microRNA-155 formatted siRNAs against human IGF2BP3 and subcloned them into pHAGE6 based lentiviral vectors. Our preliminary data demonstrates that these vectors are capable of knocking down IGF2BP3 in the NALM6 cell line. In addition, cells with knockdown showed a dramatic decrease in their growth rates, as measured by the MTS assay.The IGF-2 paracrine signaling system is thought to be important in the maintenance of HSCs as well as in lymphocyte development. We separated different precursors of B-cells (Hardy fractions) from murine bone marrow using FACS and measured the mRNA expression of CD44 and IGF2BP3 in these different subsets. The expression of both these genes correlated well with each other and showed a dynamic expression pattern with the highest expression seen in the Hardy Fraction C (late pro-B cells). This indicates that the IGF2BP3/CD44 axis might play a role in regulating normal B-cell development and this may be dysregulated in MLL-translocated B-ALL.To examine whether IGF2BP3 overexpression causes leukemia, we cloned the murine and human IGF2BP3 coding regions in a murine retroviral expression vector, MIG (MSCV-IGF2BP3-IRES-GFP). Retroviral packaging was done using 293T cell line, virus was collected and used to infect 7Oz/3, a murine pre-B ALL cell line. Western blot and qPCR confirmed overexpression of IGF2BP3. We have infected bone marrow cells from CD 45.2 positive wild type donor mice with the virus and transferred them into irradiated CD 45.1 recipient mice. We have confirmed engraftment in these mice using flowcytometry for CD 45.1/2 and are presently following the mice for the development of leukemia.In summary, IGF2BP3 is dysregulated in MLL-rearranged leukemia, and its knockdown can cause decreased growth rates in B-ALL cell lines. The current study explores whether IGF2BP3 is oncogenic and the mechanisms of action of IGF2BP3 in B-cell development and neoplasia. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Status Of CEBPA Promoter and Expression Of CEBPα Correlates With Molecular Genetic Subtype and CD2 Aberrant Expression In B Cell Precursor ALL

CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL).Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A). [Display omitted] In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p<0.0001). Interestingly, in the only hypomethylated ETV6-RUNX1pos case we found aberrant CD2 expression on blasts, which is exceptional in ETV6-RUNX1pos ALL.We next asked whether methylation of CEBPA promoter correlates with CEBPα expression. It is generally accepted that promoter hypomethylation is often associated with increased expression of the relevant gene. Our data prove that in general, this holds true also for BCP-ALL. However, in two genetically defined subsets we observed either high expression despite hypermethylation (MLL-rearranged ALL) or low expression despite hypomethylation (mBCR-ABLpos ALL) (Figure 1B). In BCP-others hypomethylation of CEBPA promoter was significantly associated with upregulation of myeloid antigens (CD14 and/or CD33) and downregulation of B cell marker CD19 on blasts during the first weeks of the treatment (Fisher test, p=0.0009). In summaryMethylation status of CEBPA promoter correlates with genetic subtypes of BCP-ALL. The notion that hypomethylation leads to overexpression was confirmed in majority of BCP-ALLs, while in mBCR-ABLpos and MLL gene rearranged BCP-ALL it did not follow this pattern. Hypomethylation of CEBPA promoter in BCP- others correlates with CD2 expression on blasts and increased CEBPα gene expression. During the early phase of the treatment in other BCP-ALLs with hypomethylated CEBPA promoter increase of myeloid and decrease of B lymphoid markers on blasts was observed.Supported by: GACR P301/10/1877, GACR P304/12/2214, GAUK 914613, UNCE 204012, NT13462, NT12397- 4, project for conceptual development of research organization (Ministry of Health, CZ) 00064203, the FACS Aria instrument was supported by EU-Prague project CZ.2.16/3.1.00/24022 Disclosures:No relevant conflicts of interest to declare.