bluuebunny/crossref_metadata_2025 huggingface dataset:
Lyn Is an Important Component of the Signal Transduction Pathway Specific to FLT3/ITD and Can Be a Therapeutic Target in the Treatment of AML with FLT3/ITD.
Authors: Hayakawa Fumihiko , Okamoto Mitsunori , Abe Akihiro , Kiyoi Hitoshi , Naoe Tomoki
Published: October 2019

Fms-like tyrosine kinase 3 (FLT3) is expressed in hematopoietic progenitor cells. An internal tandem duplication (ITD) of the FLT3 juxtamembrane domain (FLT3/ITD) is the most frequent mutation in human adult acute myeloid leukemia (AML). Being transfected into IL-3 dependent cell line, 32D, FLT3/ITD causes constitutive activation of FLT3 itself and its downstream signal components, which leads to IL-3 independent cell growth. In particular, STAT5 activation is thought to be a key signaling event since it is induced only by FLT3/ITD not by wild type FLT3. However the signal component which mediates FLT3/ITD to STAT5 is yet to be clarified. In the present study, we showed the mutant- specific association of FLT3/ITD with Lyn, which led to the phosphorylation of Lyn in vivo. We also demonstrated that Lyn directly bound to FLT3 through its SH2 domain in vitro and that the association depended on tyrosyl-phosphorylation at juxtamembrane domain of FLT3. We revealed the critical role of Lyn for STAT5 activation and the autonomous cell growth in FLT3/ITD transfectant of 32D by using anti-Lyn siRNA and the Src family kinase inhibitor PP2. More importantly, we demonstrated successful treatment of FLT3/ITD induced tumors with PP2 in the mice model. These results demonstrate that Lyn is a critical component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD.
High Expression of ENPP2/Autotaxin in Patients with FLT3-ITD Positive Acute Myeloid Leukemia.
Authors: Ortlepp Claudia , Steudel Christine , Koch Sina , Ryser Martin , Ehninger Gerhard , Thiede Christian
Published: October 2019

The Fms-like tyrosine kinase 3 (FLT-3) receptor tyrosine kinase acts as an important player in controlling the growth and differentiation of haematopoietic progenitor cells. It was shown that activating mutations of FLT-3 are involved in the pathogenesis of acute myeloid leukemia (AML). The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with poorer prognosis. To further understand the factors involved, we performed microarray analysis to identify genes specifically upregulated in cases with FLT3-ITD mutations. One gene we identified was ENPP2/Autotaxin (ENPP2/ATX). The ENPP2/ATX protein acts as a lysophospholipase D through generating lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). These two bioactive small lipids have crucial functions in cell migration and proliferation and act via G-protein coupled receptors. ENPP2/ATX has been associated with tumor cell migration in several solid tumors, expression has not been reported in myeloid cells before. The aim of the current study was to investigate the expression pattern of ENPP2/ATX in normal haematopoiesis and leukemia as well as its role in the pathogenesis of AML. Real-Time PCR analysis of 215 primary AML samples from all major cytogenetic risk groups confirmed a significant upregulation in FLT3-ITD patients. High expression was also found in cases with complex karyotype and −5 and −7, but not in cases with good risk cytogenetics. To explore the function of ENPP2/ATX in the haematopoietic system, we stably overexpressed two different splice variants of ENPP2/ATX in several leukemic cell lines. The expression was detected at both, mRNA and protein level by RT-PCR and Western blot analyses, respectively. The lysophospholipase D activity of the protein could be measured by a functional assay using an artificial substrate for ENPP2/ATX. Transwell migration assays showed an increased migration capacity of cells with high expression of ENPP2, which indicates that the overexpressed protein is functional. Based on our results, we suggest that ENPP2/ATX is involved in leukemic cell migration. Our findings point to a close link between ENPP2/ATX and FLT3 in the pathogenesis of AML. Current work focuses on its role in cell survival and prognosis.
Common Leukemic Signaling Pathways Identified by Comparative Analysis of GM-CSF and FLT3 Activated Receptor Mutations.
Authors: D’Andrea Richard , Perugini Michelle , Kok Chung , Wilkinson Christopher , Brown Anna , Gonda Thomas
Published: October 2019

As a strategy to identify key targets in myeloid leukemia we examined directly the overlap in signaling and downstream events induced by several activated receptor mutants associated with AML. We compared the events induced by a leukemic mutant (V449E) of the GM-CSF receptor with events associated with the activating FLT3 internal tandem duplication mutant (FLT3-ITD) and the D835Y kinase domain mutant (FLT3-TKD). Receptor mutants were introduced by retroviral transduction into the FDB-1 bi-potential myeloid cell line model (McCormack MP et al., Blood.95:120–127, 2000). We used biochemical analysis and pathway inhibitors to demonstrate that the GMR V449E mutant selectively activates the JAK2-STAT5 and p44/42 MAPK pathways which are central to the ability of this mutant to confer continued growth and arrested differentiation in the absence of growth factor. Like the GMR V449E mutant, the FLT3-ITD and -TKD mutants arrest granulocyte-macrophage differentiation and support continued growth in the absence of added growth factor. For both of these mutants we also observed constitutive JAK2 and STAT5 phosphorylation and sensitivity to a selective JAK2 inhibitor (JAK2 Inhibitor II, Merck) suggesting a potential key role for JAK2 in signalling from FLT3 activating mutants. Both FLT3 mutants activated the p44/42 MAPK pathway, resulted in Akt phosphorylation and were sensitive to PI3-kinase and MEK inhibitors. As activation of the p44/42 MAPK pathway is common to all mutants, and has recently been associated with suppressed differentiation in AML (Radomska HS et al, J Exp Med. 203:371–381, 2006), we used the selective MEK inhibitor, U0126 (Merck), to investigate the contribution of this pathway to maintenance of the differentiation block. Treatment of factor-independent FDB-1 cells expressing the GMR V449E mutant, or either FLT3 mutant, with U0126 for 48 hours induced morphological differentiation suggesting that the sustained activation of p44/42 MAPK contributes to maintaining the arrest in myeloid differentiation. To explore further the overlapping nature of signalling by the GMRV449E and FLT3-ITD activated mutants we utilised the Wilcoxan rank sum test to perform gene-set enrichment analysis. Analysis of 119 FLT3-ITD associated genes (126 probes) (Mizuki M et al, Blood101: 3164–3173, 2003) revealed that this gene-set shows evidence of differential expression in response to GMR V449E signaling (p=2.8x10−7). In particular, we identify the tumor suppressor gene, Gadd45α, as a common gene, down-regulated in response to signalling via the FLT3-ITD, FLT3-TKD and GMR V449E activated receptors. Analysis of AML gene expression data (Valk PJ et al, New Engl. J. Med: 350:1617–1628, 2004) indicates that activation of Gadd45α maybe suppressed by multiple pathways in AML blasts.
Antioxidants Improve Factor VIII Secretion in the Liver by Preventing Oxidative Stress, Activation of the Unfolded Protein Response, and Apoptosis.
Authors: Malhotra Jyoti D. , Zhang Kezhong , Miao Hongzi , Pipe Steven W. , Kaufman Randal J.
Published: October 2019

Factor V (FV) and Factor VIII (FVIII) are homologous glycoproteins that provide essential functions in hemostasis. Previous studies demonstrated that compared to FV, the FVIII polypeptide folds inefficiently, accumulates in the endoplasmic reticulum (ER) and activates the Unfolded Protein Response (UPR). The UPR, mediated by the proximal sensors PERK and IRE1, is a signaling pathway leading to either adaptive survival or apoptotic demise upon accumulation of unfolded proteins in the ER. Here we show that FVIII expression in hepatocytes in vivo mediated by hydrodynamic tail-vein injection of plasmid DNA into fviii −/ − mice produces between 0.5 to 1.0 Unit/ml of FVIII in the plasma. This level of FVIII expression activated PERK and IRE1 to signal the UPR and induced both markers of oxidative stress and apoptosis in hepatocytes. In contrast, injection of DNA encoding either FV or a FVIII molecule engineered for improved secretion did not induce oxidative stress, activate the UPR or induce apoptosis. Gene expression analysis demonstrated that FVIII expression induced expression of the proapoptotic transcription factor CHOP. Injection of FVIII expression plasmids into chop−/ − mice did not induce oxidative stress or apoptosis, indicating that these stress responses require CHOP. Feeding of mice with the lipid soluble antioxidant, butylated hydroxy anisole (BHA), reduced oxidative stress and apoptosis and increased expression of FVIII in the plasma, demonstrating a pivotal role of oxidative stress in limiting FVIII expression and triggering apoptosis. Finally, overexpression of the anti-apoptotic protein Bcl2 also significantly suppressed oxidative stress and apoptosis and increased FVIII expression. The results demonstrate that 1) misfolding of an ER luminal protein, FVIII, is sufficient to induce oxidative stress and apoptosis in vivo, 2) oxidative stress limits protein secretion and activates apoptosis through a mechanism that requires CHOP, and 3) intervention to prevent oxidative stress by antioxidant feeding or Bcl2 overexpression preserves ER function, improves secretion and prevents apoptosis. The findings raise the possibility to treat diseases of protein misfolding, such as certain mutations that cause hemophilia A, by treatment with antioxidants.
Effects of Initial Treatment on Survival among Elderly AML Patients: Findings from the SEER-Medicare Database.
Authors: Menzin Joseph , Boulanger Luke , Karsten Verena , Cahill Ann L. , Earle Craig E.
Published: October 2019

Background: Acute myeloid leukemia (AML) is the most common type of leukemia among adults in the U.S. However, data on the effects of treatment on survival for large numbers of AML patients in clinical practice are limited.

Methods: Using the linked SEER-Medicare database, we evaluated mortality for AML patients 65+ years of age diagnosed between 1999 and 2002 who received chemotherapy versus no chemotherapy. Patients were followed from diagnosis until death or the database cut-off (December 31, 2003). The presence of chemotherapy treatment was identified based on procedure codes and diagnosis-related groups. The two study cohorts were matched on age, sex, comorbidity (Deyo adaptation of the Charlson Comorbidity Index), geographical region, teaching hospital status, and myelodysplastic syndrome (MDS) in the prior 12 months. Mortality rates were estimated via the Kaplan-Meier technique. Subgroup analyses were performed by level of comorbidity, age group, and teaching hospital status.

Results: A total of 3,317 elderly patients with AML who were eligible for fee-for-service Medicare coverage were identified. Of these, 1,193 (36%) received chemotherapy and 2,124 did not receive treatment. Treated patients were found to be younger and had fewer comorbidities compared to untreated patients. After matching, there were 888 patients in both cohorts. Mean age was 75 years and 59% were male. Fifty-three percent of patients received care at teaching hospitals. The median Charlson score was 1.0 and 24% were diagnosed with prior MDS. Median survival was 4.4 months longer for the treated cohort than for the untreated cohort (6.1 versus 1.7 months; P<0.0001) (see Figure).

Conclusions: This large observational study of elderly patients with AML illustrates that chemotherapy treatment is associated with improved survival. This survival gain is slightly less than that observed in other population-based AML studies that did not match on key prognostic factors, such as age and comorbidity levels.

Survival rates for treated and untreated patients Survival rates for treated and untreated patients
Treatment of AML in the Elderly: Who Is “Fit” for Intensive Chemotherapy?.
Authors: Hills Robert K. , Kell William J. , Burnett Alan K.
Published: October 2019

Currently, increasing attention is being focused on the older patient with AML who is deemed “unfit” for intensive chemotherapy. Indeed a recent population-based study (Juliusson et al, Leukemia 2006 (20), 42–7) showed that only around one half of patients between the ages of 70 and 79 were offered remission induction chemotherapy. Typically the unfit patients are treated with supportive care, low doses of cytosine arabinoside (Ara-C) or with investigational agents. However, there is no strict definition of what constitutes a patient unfit for intensive chemotherapy. The NCRI AML14 trial offered patients the choice of entering an intensive chemotherapy randomisation (where patients received daunorubicin/Ara-C based therapy, n=1273), or a non-intensive randomisation between low dose Ara-C and hydroxyurea (n=217). A randomisation between intensive and non-intensive chemotherapy was attempted but recruited only 8 patients, indicating that clinicians did not appear to be uncertain about which patients should receive which type of treatment. In order to identify which factors were significantly associated with the decision to enter either the intensive or non-intensive approach we performed analyses of the AML14 data. The two principal factors associated with fitness for intensive chemotherapy were age and performance status (both p<0.0001). Other factors influencing the decision were presence of comorbidities (p=0.007), diagnosis (de novo v secondary, p=0.01), lower weight (p=0.04) and heart disease (p=0.04). However, even after adjusting for these factors, clinician was a highly significant predictive variable (p<0.0001), and this significance was retained even if analyses were restricted to those doctors who had entered patients into both parts of the trial. While it is unsurprising that patients deemed unfit for intensive chemotherapy are older, less fit, thinner and with more comorbidities, the high level of significance of clinician as a variable indicates that this characterisation is insufficient. It is likely that clinician is a surrogate variable for other factors currently not routinely collected, although there is also a suggestion that clinicians may not be consistent in who receives intensive treatment. Comparisons of patients treated intensively and those treated non-intensively, even when stratified for prognostic factors, tend to show that intensively treated patients perform better (Wheatley et al., Blood 2005 (106(11)) 199a (Abstract 674)) but it is unclear whether even this stratified comparison is confounded.

Conclusion: This study defines factors which differ between patients allocated to an intensive or non-intensive approach. However we identify the clear influence of which physician is involved in the choice as an independent factor. To identify factors (which may be components of the physician’s choice) reliably a more comprehensive index is needed for which a prototype will be presented.
Effectiveness of Clofarabine in Elderly AML Patients with Adverse Cytogenetics Unfit for Intensive Chemotherapy.
Authors: Burnett Alan K. , Baccarani M. , Johnson Peter , Yin John , Saunders Andrew , Russell Nigel , Hills Robert
Published: October 2019

Outcomes for elderly patients with AML are almost uniformly poor, and it has been shown (Wheatley et al., Blood2005, 106(11) 199a) that patients with adverse cytogenetics have a particularly poor prognosis. Additionally, a large proportion of such patients are considered not to be fit for intensive chemotherapy (Juliusson et al, Leukemia, 2006 (20): 42–7), and given best supportive care, or low-dose Ara-C. The NCRI AML14 trial observed no remissions in 18 patients with adverse cytogenetics treated with Ara-C, and no remissions in 20 patients treated with hydroxyurea. We present here a combined analysis of the BIOV-121 Study, and the UWCM-0001 study. Both studies were for untreated patients over 65 years of age who were not considered fit for chemotherapy. The treatment comprised clofarabine 30mg/m2 on days 1–5 repeated after 28–42 days for up to 3 courses. The primary endpoints were overall response (CR, CRi or PR) and safety.

Patients: A total of 95 patients were recruited, of whom 95 received clofarabine 30mg/m2. Twenty-six patients (27%) had adverse cytogenetics.

Results: 10/26 patients (38%) achieved either CR or CRi. With a median follow-up of 10 months (range 2–17 months), 1 year survival is 20%, with 19/26 patients having died. At most recent follow-up, 4/10 patients achieving CR or CRi are still alive in remission.

Discussion: The remission rate of 38% seen with clofarabine compares favourably, not only with traditional non-intensive approaches (which have failed to induce any remissions in similar sized cohorts), but also to the 42% remission rate seen in patients treated with intensive daunorubicin/Ara-C based chemotherapy in the NCRI AML14 trial. One year survival rates are also encouraging, at 20% compared to 0%, 5% and 23% for patients treated with Ara-C, supportive care, and intensive chemotherapy respectively. Exploratory comparisons of survival between the 30mg clofarabine patients and the three treatment regimens give highly significant survival advantages compared to Ara-C and HU (p=0.0001, p=0.004 respectively) and no significant difference between clofarabine and DA (p=1.0), although confidence intervals in this case are wide.

Conclusions: Clofarabine has the ability to induce remissions in patients with adverse cytogenetics, unlike Ara-C and supportive care, and survival in this group is improved. Remission rates and survival are similar to those seen for patients treated with intensive chemotherapy. Clofarabine is clearly worthy of further investigation, and may provide an important treatment option for high risk patients with AML.
Long Term Data on Anthracycline Cardiotoxicity in 547 Children with AML.
Authors: Creutzig Ursula , Diekamp Sylke , Zimmermann Martin , Reinhardt Dirk
Published: October 2019

Anthracyclines are effective antineoplastic drugs in acute mylogenous leukemia (AML) and dose intensity and cumulative doses are especially important. However, the use of anthracyclines is limited by cardiotoxicity, which occurs already at 300 mg/m2 cumulative doses (given as daunorubicin dosage) in children. To evaluate anthracycline-associated cardiomyopathy in pediatric AML-patients, the incidence of late clinical and subclinical cardiotoxicity (using echocardiography) was analysed in studies AML-BFM 93 and 98. Out of a total of 1,207 patients under 18 years, 547 (45%) patients were eligible for the analysis of late cardiotoxicity - de novo AML: 470 of 1,010 (46%), AML-Down syndrome: 69 of 121 (57%), secondary AML 8 of 76 (11%) -, median follow-up 5.3 years (0.8–11.7 years) after diagnosis. The cumulative dose of anthracyclines was risk-adapted between 300 and 450 mg/m2 or even higher in patients with secondary AML.

Results: Late cardiomyopathy was seen in 16 patients, cumulative incidence (CI) after 11 years 5±1% (de novo patients: 4±1%), including 4 of 38 patients (10.5%) who suffered already from early cardiomyopathy. Nine of these 16 patients (CI 2.5±1%) showed clinical symptoms, with persistent abnormal shortening fraction in 5 of them, which led to death in one AML-Down syndrome patient. Late subclinical cardiomyopathy occurred temporarily in 7 patients. Late clinical cardiomyopathy mainly effected patients with a second anthracycline therapy (3 of 8 with AML as secondary malignancy) and those with early cardiotoxicity. Using anthracyclines risk-adapted or administering long-term infusion might have contributed to the low rate of cardiomyopathy in AML de novo patients.
NPM Mutations and Not FLT3 Mutations Are a Potential Marker for Monitoring Minimal Residual Disease in Acute Myeloid Leukemia.
Authors: Palmisano Michela , Grafone Tiziana , Ottaviani Emanuela , Testoni Nicoletta , Paolini Stefania , Papayannidis Cristina , Rondoni Michela , Baccarani Michele , Martinelli Giovanni
Published: October 2019

Nucleophosmin (NPM) is a multifunctional phosphoprotein that acts as nucleocytoplasmic shuttling protein, with tumour suppressor and oncogenic functions. Recently, NPM mutations have been found in a subset of adults with de novo acute myeloid leukemia (AML). These mutations occur in the last coding exon (exon 12), causing a frameshift and the formation of novel C-termini. The abnormal mutated NPM protein shows aberrant cytoplasmic localization and is frequently associated with FLT3 mutation.

These observations provide basis for studies of the pathogenesis in AML. We did sequential analysis on patient samples during the clinical course to investigate the stability and pathogenetic role of NPM mutation in AML and the association with FLT3 mutations. The NPM mutations were determined by D-HPLC analysis; samples exhibiting an abnormal D-HPLC profile were confirmed by direct sequencing. We investigated 28 patients of de novo AML. Eleven samples were sequenced since they showed an heteroduplex D-HPLC profile. Type A mutation (960_963dupTCTG) was the commonest observed change, occurring in 9/11 samples, followed by type B mutation (960_963insCATG) in 1 case and type D mutation (960_963insCCTG) in 1 case. Furthermore, we observed that 5/11 patients harboring NPM mutation presented also mutant FLT3 at diagnosis. Analyzing NPM mutations during progression of disease, we observed that NPM mutation disappeared at complete remission and the same mutation reappeared at relapse. No differences were found in wild type NPM.

Instead, we found a modification of FLT3 status associated to evolution of disease in 7/28 patients: 2 patients lost the mutation at relapse, 4 patients acquired the mutation at relapse and 1 patient modified the mutation from D835 to ITD (Table 1). Together these results suggest that NPM mutations and not FLT3 mutations may have utility as a potential marker for monitoring minimal residual disease. Studies on the biological effects of NPM mutations will contribute to disclose the role of NPM mutations in the pathogenesis of AML and their interactions with other genetic alterations such as FLT3.
Sonic Hedgehog Signaling in Mantle Cell Lymphoma.
Authors: Hegde Ganapati V. , Emanuel Katy , Joshi Avadhut D. , Munger Corey M. , Weisenburger Dennis D. , Vose Julie M. , Joshi Shantaram S.
Published: October 2019

Mantle cell lymphoma (MCL) is associated with poor clinical outcome among all the B-cell malignancies. MCL cells have a characteristic phenotype including IgM+, IgD+, CD5+, CD10−, CD19+, CD20+, Bcl2+, CD23−, CD24+, and have a (11:14) (q13:q32) translocation, involving the Bcl-1 locus and the IgH heavy chain joining region, resulting in up-regulation of Bcl-1/PRAD-1 with increased Cyclin D1 expression. In spite of our understanding of B cell development, we do not have effective therapeutic approach to treat MCL. This warrants an immediate understanding of the biology MCL cells, and key signaling pathways underlying the disease progression. Further, these key signaling pathways involved in MCL could be specifically targeted, which may have potential therapeutic value.

Emerging evidences have revealed that sonic hedgehog (Shh) signaling promotes tumor cell proliferation, and protects them from apoptosis in different cancers such as pancreatic cancer, prostate cancer, gastric cancer, medulloblastoma, basal cell carcinoma, breast cancer, etc. In addition, targeting the Shh signaling has been shown as a potential therapeutic approach to treat some of these cancers. However, the role of Shh signaling has not been reported in any of B cell malignancies including MCL. Therefore, we studied the status of Shh signaling molecules in the proliferation and survival of MCL cells in vitro using JVM-2, Granta-519, Jeko-1 and Z138 cell lines and human primary MCL cells. Our results demonstrate that the molecules involved in the Shh signaling like patched and smoothened receptors, and the target transcription factors, GLI were over expressed in Granta-519, Jeko-1, Z-138 and JVM-2 MCL cell lines, and patients primary MCL cells compared to normal human B lymphocytes. Addition of exogenous Shh increased the proliferation of JVM-2 cells in vitro. There was an increased transcripts level of GLI1, BCL2 and Cyclin D1 (CCND1) in JVM-2 cells following the addition of Shh. Addition of Shh-signaling inhibitor Cyclopamine abrogated the increased proliferation and transcription of the above genes induced by Shh in JVM-2 cells (figure 1). Furthermore, the Gli2 anti-sense oligonucleotide decreased CCND1 and BCL2 transcript expression, as well as decreased the proliferation of JVM2 cells in vitro (figure 2). Taken together, these results suggest that Shh-Gli signaling may be one of the pathways that regulate the Bcl-2 and Cyclin D1 activation and thereby regulate the proliferation and apoptosis of MCL cells. Therefore, further dissecting of the Shh-Gli mediated regulation of Bcl2 signaling may have potential implications in advancing the treatment for MCL.

Figure 1: Figure 1:. Figure 2: Figure 2:.
aUPD in the Clonal Evolution of Follicular Lymphoma.
Authors: O’Shea Derville , Iqbal Sameena , Carlotti Emanuela , Davies Andrew , Chaplin Tracy , Matthews Janet , Raghavan Manoj , Norton Andrew , Lister T. Andrew , Young Bryan D. , Fitzgibbon Jude
Published: October 2019

The use of Single Nucleotide Polymorphism (SNP) array profiling has uncovered extensive regions of acquired uniparental disomy (aUPD) in the cancer genome that go undetected using cytogenetics or array-CGH platforms. These regions usually arise by mitotic recombination and can render a cell homozygous for a pre-existing abnormality. SNP array analysis was performed using the Affymetrix 10K Gene-chip mapping array on DNA extracted from a series of Follicular Lymphoma (FL) lymph nodes biopsies from 42 patients, taken at time of diagnosis (n=20), progression (n=16) and transformation (t-FL) (n=32), and the t(14;18) positive lymphoma cell lines DoHH2 and RL 2261. Analysis was performed using the genome oriented laboratory file system, a software package designed to interpret SNP data. The criteria of > 96% homozygosity in at least 50 contiguous SNPs was found to detect no abnormalities in 24 normal remission bone marrows and was therefore adopted for the detection of abnormal runs of homozygosity. Abnormalities were detected in 53/68 primary specimens; these were non-random with recurring sites of aUPD on several chromosomes including 6p, 9p, 12q and 17p. This panel included 26 paired FL and t-FL samples; in 9 FL cases regions of homozygosity were identified which were not present in the subsequent t-FL sample, suggesting that t-FL may arise in a proportion of patients by a mechanism other than a process of direct clonal evolution. Homozygosity of 9p and 17p was seen primarily in the transformation samples and in three cases rendered the cell homozygous for a pre-existing mutation of either CDKN2A or TP53. Thus mutation precedes mitotic recombination which leads to the removal of the remaining wild-type allele. Nine out of 42 patients studied (21%) have aUPD of chromosome 6p. This appears to be an early event in lymphomagenesis as it was present in both FL and t-FL samples obtained from the same patient. The sites of mitotic recombination cluster in a region immediately proximal to the MHC complex at 6p21-12 in 8/9 cases. Mutations in CCND3, CDKN1A, and two translocation partners of BCL6: SRP20 and HIST1H4I, which are all located within or just distal of this cluster, were excluded by direct sequence analysis. Loss of MHC class II expression has been frequently observed in Diffuse Large B-cell lymphoma and a selective advantage gained by the lymphoma cell here would have implications in tumour surveillance. This study highlights the frequency of aUPD in follicular lymphoma and ongoing studies will elucidate the selective basis of a UPD at this location in the pathogenesis of lymphoma.
PVRL2 Is Translocated to the TCRA Locus in t(14;19)(q11;13) Positive Peripheral T-Cell Lymphoma.
Authors: Almire Carole , Ruminy Philippe , Bertrand Philippe , Wlodarska Iwona , Subero Jose I. Martin , Siebert Reiner , Tilly Herve , Bastard Christian
Published: October 2019

T-cell non-Hodgkin lymphomas (NHLs) are uncommon malignancies that represent approximately 15% of aggressive lymphomas in Western countries. Contrasting with B-cell lymphomas, very few recurrent chromosomal abnormalities have been identified. Nevertheless, the TCR loci and preferentially the TCRAD at chromosome band 14q11 could be involved in up to 15% of cases. We recently reported a novel and recurrent t(14;19)(q11;q13) chromosomal translocation in peripheral T-cell lymphoma (PTCL). FISH analysis performed in three cases suggested an implication of the TCRAD locus at 14q11 and of BCL3 at 19q13. We now report the molecular cloning of the genomic junctions for these three patients. Sequence analysis confirmed the involvement of the TCRAD on 14q11 and assessed the breakpoint’s localisation to the TCRA variable region for all cases. In the first, the chromosomal break occurred between the J30 and the J31 segments while in the second it was in the J58 segment. Thus, both translocations probably occurred after an illegitimate V(D)J recombination. Remarkably, for the third patient, junctions with the deltaRec and psi(J)alpha elements were identified on the der(14) and der(19) respectively. Consequently this translocation probably results from an illegitimate deltarec-Psi(J)alpha rearrangement (TCRD deletion) during thymopoiesis before Valpha-Jalpha rearrangements. On chromosome 19, our results indicated a new clustering of breakpoints outside the region documented in t(14;19)(q32;q13) in chronic lymphocytic leukemia. Remarkably, all three breaks occurred ~130kb downstream from BCL3, in the PVRL2 gene. Consequently, the TCRA locus enhancer is juxtaposed to PVRL2 and BCL3 on the derivative chromosome 19. For two patients, mRNA expression levels were investigated by real time RT-PCR experiments, which confirmed a high expression of both PVRL2 and BCL3. In conclusion, we identified PVRL2 as the recurrent translocation partner of the TCRA locus in PTCL. Whether this clustering results from an important instability of the PVRL2 gene during thymopoiesis remains to be investigated. The PVRL2 overexpression itself may be an important step for T-cell lymphomagenesis.
FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] Propane 1, 3-diol hydrochloride), Mediates Cytotoxicity through Caspase Independent and Protein Phosphatase 2A Dependent Mechanisms in Chronic Lymphocytic Leukemia and Lymphoblastic Leukemia/Lymphoma.
Authors: Liu Qing , Zhao Xiaobin , Frissora Frank , Ma Yihui , Santhanam Ramasamy , Perrotti Danilo , Chen Ching-Shih , Dalton James T. , Byrd John C. , Muthusamy Natarajan
Published: October 2019

FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] propane 1, 3-diol hydrochloride) is a synthetic compound produced by modification of a natural immunosuppressant, ISP-1. It is an immunosuppressive agent that is being developed to prevent organ transplant rejection. Recent studies indicate additional role for FTY720 in inducing cell apoptosis. We demonstrate here a novel mechanism by which FTY720 mediates cytotoxic effects in cell lines representing different B cell malignancies and primary B cells from chronic lymphocytic leukemia (CLL) patients. FTY720 induced apoptosis as detected by annexin V/ propidium iodide staining in representative B cell lines and CLL patient derived CD19+ B cells in time and dose dependent manner (p<0.0001, untreated vs 10mM-treated CLL cells, n=15). In contrast to previous reports in T cell lines, FTY720 induced cytotoxicity in Raji cell line and primary CLL cells is independent of activation of caspase 3, 8 and 9 or poly-ADP ribose polymerase cleavage. Further, pan-caspase inhibitor Z-VAD-fmk rescued these cells from fludarabine but not FTY720 induced apoptosis (p=0.001 fludarabine vs fludarabine+z-VAD-fmk; p=0.99 FTY720 vs FTY720+z-VAD-fmk, n=5). Over-expression of Bcl-2 failed to protect transformed B-cells from FTY720 induced apoptosis suggesting Bcl-2 independent cytotoxic effect. Interestingly, FTY720 induced consistent increase in protein phosphatase 2a (PP2a) activity and concentrations of okadaic acid that inhibited the FTY720-induced PP2A activity also resulted in inhibition of FTY720-mediated cytotoxicity in B cell lines and primary CLL cells, indicating a role for PP2A activation in FTY720 induced cytotoxicity. Consistent with its activation of PP2A, FTY720 induced dephosphorylation of of Erk1/2 in CLL B cells. Further, FTY720 treatment resulted in significant in-vivo therapeutic efficacy associated with prolonged survival in a xenograft SCID mouse model of disseminated B cell lymphoma/leukemia (median survival time for FTY720 treated mice was 47 days (95 %CI 39–53) compared to 18 days in placebo controls (95% CI 17–19) P<0.0001-FTY720 vs placebo). These results provide first evidence for a PP2A dependent and caspase independent cytotoxicity of FTY720 in B cells. The novel caspase and Bcl-2 independent mechanism of cytotoxicity concurrent with identification of PP2a activation as a surrogate marker of cell killing provide further justification for clinical development of this agent in lymphoid leukemia.
Ethacrynic Acid Can Inhibit Wnt/β-Catenin Signaling and Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.
Authors: Liu Jerry X. , Endo Tomoyuki , Kipps Thomas J. , Dennis Carson A. , Desheng Lu
Published: October 2019

Chronic lymphocytic leukemia (CLL) is caused by an abnormal balance in signals for survival and cell death. Recently, CLL cells are shown to have an activated Wnt/β-catenin pathway, which may contribute to the apoptosis-resistant state characteristic of this disease. Accordingly, inhibitors of the Wnt/β-catenin pathway may compromise the viability of CLL cells in vitro and in vivo. To identify potential inhibitors of the Wnt/β-catenin pathway, we screened the Gen-Plus drug library using a Wnt/β-catenin TOPflash reporter plasmid in HEK293 cells. Although 99% of the 960 drugs screened with this assay, including many clinically used alkylating agents and anti-metabolites, did not inhibit the Wnt/β-catenin pathway, ethacrynic acid (EA), a once commonly used diuretic agent, displayed potent inhibition of Wnt/β-catenin signaling. EA has an unsaturated lactone nucleophile that can react with sulfhydryl groups in proteins. N-acetyl-L-cysteine (NAC), but not other anti-oxidants, prevented EA from inhibiting the trans-activation function of β-catenin. Treatment of CLL cells with EA caused a time-dependent decrease in protein levels of the Wnt/β-catenin regulated LEF-1 transcription factor, which is overexpressed in CLL cells, compared to normal blood lymphocytes. Treatment with EA also caused a concentration dependent decrease in cyclin D1 and fibronectin levels in CLL cells as detected by Q-PCR. Collectively, these results suggest that EA can inhibit Wnt/β-catenin signaling in CLL cells, and this effect is mediated by the ability of EA to modify sulfhydryl groups on key proteins, rather than by non-specific nucleophilic attack or increased cellular oxidative stress alone. Cell viability assays showed that EA induced cell death in primary CLL cells with a mean IC50 of 3 uM, a concentration achieved in previous clinical trials with the drug in patients with solid tumors. EA at 3 uM also enhanced by 6–10 fold the cytotoxicity of fludarabine in CLL cells from both fludarabine sensitive and resistant patients. At the same concentration, EA did not cause significant cell death of normal blood mononuclear cells. These results, therefore, suggest the therapeutic potential of EA in CLL, based on a novel mechanism of targeting the Wnt/β-catenin signaling pathway.
Clinical Significance of Loss of Chromosome Y (−Y) in Patients with Chronic Myeloid Leukemia (CML): Is It Clonal Evolution?.
Authors: Jabbour Elias , Kantarjian Hagop , Ravandi-Kashani Farhad , Garcia-Manero Guillermo , O’Brien Susan , Cortes Jorge
Published: October 2019

Clonal evolution (CE), in chronic myeloid leukemia (CML), is characterized by the appearance of additional cytogenetic abnormalities in Philadelphia-positive (Ph+)-cells and is considered a criterion of accelerated phase (AP). Loss of chromosome Y (−Y) frequently occurs in male individuals with aging and has been generally not considered a clinically significant abnormality in hematologic malignancy. In CML it is most frequently not considered an abnormality representing CE assuming it lacks the prognostic implications of other abnormalities in this setting. We thus investigated the frequency and prognostic significance among 352 patients with CML in chronic phase (CP) treated with imatinib mesylate at our institution. −Y was detected along with Ph+ in 7 patients (2%): 3 at the time of diagnosis and 4 during the course of therapy, after a median of 47 months (range, 9–130 months) from diagnosis. Median age was 56 years (range, 40–75 years). Three patients had received prior interferon. Best response to imatinib was complete cytogenetic response in 2 patients (29%), partial in 1 (14%), minor in 2 and complete hematologic response in 2. All but one patient have lost their response after a median of 26 months (range, 2–55 months). In only one patient, −Y was transient and disappeared after 3 months. This patient is in ongoing molecular remission. Of the 6 patients who failed imatinib, 3 patients also failed subsequent therapies (nilotinib, dastinib, and chemotherapy, respectively) and 2 of them subsequently died of progressive disease. The other 3 patients responded to salvage therapy (combination of farnesyl transferase inhibitors and imatinib, homoharingtonine, and chemotherapy), and one of them is in molecular remission following allogeneic stem cell transplantation. The median overall survival from the appearance of the −Y has not been reached. We conclude that CE involving −Y abnormality is uncommon in patients with CML treated with imatinib but it may be associated with poor outcome and should be considered as a manifestation of clonal evolution.
Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.
Authors: Wu Ji , Meng Feng , Talpaz Moshe , Donato Nicholas J.
Published: October 2019

The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.
Apoptotic Death of Bcr-Abl-Expressing Myeloid Progenitors in Response to the m-Tor Inhibitor RAD001 (Everolimus) Is Promoted by the Nuclear Import of c-Abl.
Authors: Mancini Manuela , Zuffa Elisa , Corrado Patrizia , Brusa Gianluca , Petta Sara , Pagnotta Eleonora , Santucci Maria Alessandra
Published: October 2019

Nuclear accumulation of c-Abl protein in response to genotoxic damage addresses apoptotic cell death. The process, driven by the disruption of its binding to 14-3-3 scaffolding proteins, is independent from c-Abl catalytic state and conditional upon 14-3-3 phosphorylation by the c-jun N-terminal kinase (JNK) (Yoshida et al Nat Cell Biol 7,278,2005). Our work moved from the observation that the constitutive activation of Abl tyrosine kinase (TK) by juxtaposed Bcr sequences in p210 fusion protein Bcr-Abl precludes c-Abl nuclear import in response to gamma irradiation, advancing a role for residual c-Abl protein “loss of function” in the apoptosis-resistant phenotype of Chronic Myeloid Leukemia (CML) progenitors. In 32D cell clones transducing a temperature-conditional Bcr-Abl mutant, active p210 TK is associated with the overexpression of 14-3-3sigma mostly driven by transcriptional events involving histone H4 acetylation (but not methylation) status of the gene promoter. P210 TK inhibition in response to 24 hour exposure to imatinib mesylate (IM) is associated with an early downregulation of 14-3-3sigma followed by c-Abl nuclear import and cell commitment to apoptotic death. The two last events are further enhanced by complementary inhibition of the 14-3-3 binding site by R18 peptide, supporting that the negative impact of p210 TK on pro-apoptotic function of residual normal c-Abl arises from its effects on 14-3-3sigma. Previous studies proved that the activation of p38 MAP kinase contributes to IM effects in CML (Parmar et al, J Biol Chem279,25345,2004). Interestingly, p38 MAP kinase is also involved in tuberous sclerosis 2 gene protein (TSC2, also known as tuberin) phosphorylation and enhanced binding to 14-3-3 possibly promoting the activation of IM-compensatory signals, including m-Tor (Li et al, J Biol Chem278,13663,2003). Here we show that the m-Tor inhibitor RAD001 (everolimus; kindly provided by Novartis Pharma AG) at 100 nM concentration induces c-Abl nuclear import and apoptotic cell death in 32D cell clones expressing active p210 TK. Both events are further and significantly enhanced by combination with IM. C-Abl nuclear shuttling in response to the RAD001 and IM combination procedes from Akt and p38 MAP kinase inactivating dephosphorylation and 14-3-3 phosphorylation at serine residues critical for client protein binding including the apoptotic death effectors Bad, Bax and Ask1. In conclusion, our work supports the advantage of TK and m-Tor inhibitor association for CML treatment.
Trough Plasma Imatinib Concentrations Are Associated with Both Cytogenetic and Molecular Responses to Standard-Dose Imatinib in Chronic Myeloid Leukemia.
Authors: Picard Stephane , Titier Karine , Etienne Gabriel , Lassalle Regis , Marit Gerald , Reiffers Josy , Begaud Bernard , Moore Nicholas , Molimard Mathieu , Mahon Francois-Xavier
Published: October 2019

BACKGROUND

Variable responses to imatinib, in the treatment of chronic myeloid leukemia (CML), are incompletely understood. Previous studies focused on cellular mechanisms of resistance to imatinib. Another hypothesis for variable response lies in pharmacokinetic variability that may reduce drug exposure in patients receiving imatinib.

METHODS

Using high-performance liquid chromatography-tandem mass spectrometry, we assessed trough plasma imatinib concentrations in CML patients who responded or not to standard-dose imatinib, after at least one year’s treatment. Complete cytogenetic response (CCR) was defined as 0 percent Philadelphia-chromosome-positive cells in bone marrow. Major molecular response (MMR) was defined as a 3 logarithm reduction of BCR-ABL transcripts, quantified from peripheral blood using real-time quantitative reverse-transcriptase polymerase chain reaction.

RESULTS

Sixty-eight patients were included. Mean trough plasma imatinib concentrations were significantly higher in the group with CCR (56 patients) than in the group without (P=0.02). Mean (±SD) trough plasma imatinib concentrations were significantly higher in the group with MMR (34 patients) than in the group without (1452.1±649.1 ng per milliliter vs. 869.3±427.5 ng per milliliter, P<0.001) whereas there was no difference in the imatinib daily dose. For trough plasma imatinib concentrations and their discrimination potential for MMR, the area under receiver-operating characteristic curve was 0.775, with best sensitivity (76.5 percent) and specificity (70.6 percent) at a plasma threshold of 1002 ng per milliliter.

CONCLUSIONS

Monitoring of plasma imatinib concentrations may become part of standard management of CML patients, or should at the very least be checked in the case of treatment failure or suboptimal response.