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Emergence of clones carrying point mutations in the BCR‐ABL1 kinase domain (KD) is a common mechanism of resistance to tyrosine kinase inhibitor (TKI)‐based therapies in Philadelphia chromosome‐positive (Ph+) acute lymphoblastic leukemia (ALL). Sanger sequencing (SS) is the most frequently used method for diagnostic BCR‐ABL1 KD mutation screening, but it has some limitations—it is poorly sensitive and cannot robustly identify compound mutations. Next‐generation sequencing (NGS) may overcome these problems. NSG is increasingly available and has the potential to become the method of choice for diagnostic BCR‐ABL1 KD mutation screening. A group discussion within an ad hoc constituted Panel of Experts has produced a series of consensus‐based statements on the potential value of NGS testing before and during first‐line TKI‐based treatment, in relapsed/refractory cases, before and after allo‐stem cell transplantation, and on how NGS results may impact on therapeutic decisions. A set of minimal technical and methodological requirements for the analysis and the reporting of results has also been defined. The proposals herein reported may be used to guide the practical use of NGS for BCR‐ABL1 KD mutation testing in Ph+ ALL., This consensus paper presents the results of an initiative by an expert panel to define a set of indications for the practical use of next‐generation sequencing for BCR‐ABL1 kinase domain mutation screening in Philadelphia‐positive acute lymphoblastic leukemia patients receiving tyrosine kinase inhibitor‐based therapies. Minimal technical and methodological requirements for the analysis and the reporting of results have also been proposed.

Acute Lymphoblastic Leukemia (ALL) is the most frequent cancer in pediatric population. Although the treatment has improved and almost 85% of the children are cured about 20% suffer relapse, therefore finding molecules that participate in the pathogenesis of the disease for the identification of relapse and patients at risk is an urgent unmet need. Class I myosins are molecular motors involved in membrane tension, endocytosis, phagocytosis and cell migration and recently they have been shown important for development and aggressiveness of diverse cancer types, however Myo1g an hematopoietic specific myosin has not been studied in cancer so far. We evaluated the expression of Myo1g by qRT-PCR, Immunocytochemistry and Immunofluorescence in a cohort of 133 ALL patients and correlated the expression at diagnosis and after treatment with clinical features and treatment outcomes. We found high expression levels of Myo1g in Peripheral Blood Mononuclear Cells (PBMCs) from patients with ALL at diagnosis and those levels decreased after complete remission; furthermore, we found an increase in Myo1g expression on patients with 9:22 translocation and those who relapse. This study show that Myo1g is over expressed in ALL and that may participate in the pathogenesis of the disease specially in high-risk patients.

Inherited predispositions to acute lymphoblastic leukemia have been well investigated in pediatric patients, but studies on adults, particularly Chinese patients, are limited. In this study, we conducted a genome-wide association study in 466 all-age Chinese patients with Acute lymphoblastic leukemia (ALL) and 1,466 non-ALL controls to estimate the impact of age on ALL susceptibility in the Chinese population. Among the 17 reported loci, 8 have been validated in pediatric and 1 in adult patients. The strongest association signal was identified at ARID5B locus and gradually decreased with age, while the signal at GATA3 exhibited the opposite trend and significantly impact on adult patients. With genome-wide approaches, germline variants at 2q14.3 rank as the top inherited predisposition to adult patients (e.g., rs73956024, P = 4.3 × 10-5) and separate the genetic risk of pediatric vs. adult patients (P = 3.6 × 10-6), whereas variants at 15q25.3 (e.g., rs11638062) have a similar impact on patients in different age groups (overall P = 2.9 × 10-7). Our analysis highlights the impact of age on genetic susceptibility to ALL in Chinese patients.

Summary Late relapse [>3 years from complete remission (CR)] in acute lymphoblastic leukaemia (ALL), is unusual. Data from the MRC UKALLXII/ECOG E2993 trial are presented to evaluate the incidence and characteristics of late relapse in adult ALL. Of 1,909 patients, 1,752 (92%) achieved CR and among these 757 (43·2%) relapsed; 691 (91·3%) within three years and 66 (8·7%) beyond. Among these 66 patients, median time to relapse was 47 (37–144) months. Relapse beyond three years occurred in 3·8% of all who achieved CR. The cumulative risk of relapse was 40%, 43% and 45% at three, five and ten years respectively. Out of the 1 752 patients who achieved CR, 11·7% underwent autologous and 40·6% allogeneic transplant, while in CR1. Of the autologous patients, 43·2% relapsed early and 3·4% relapsed late. However, among the allogeneic patients, 13·2% relapsed early and only 1·3% late. The five‐year overall survival from relapse was 5·8% and 20% in the early and late relapse patients respectively. In conclusion, late relapse in adults with ALL is not uncommon, and is associated with better outcome after relapse compared to early relapse.

Background: Berberine (BBR) has gained considerable attention because of its anti-tumor activity. BBR can induce apoptosis of acute lymphoblastic leukemia (ALL) cells through the MDM2/p53 pathway. However, the effects of BBR on those ALL patients with p53 deficiency remain unclear. Results: We found that BBR reduced ALL cell viability and induced apoptosis in p53-null EU-4 and p53-mutant EU-6 cells by downregulating X-linked inhibitor of apoptosis protein (XIAP), which is increased in ALL tissues and cells. BBR-induced cell apoptosis was attenuated by inhibition of XIAP that was controlled by PIM-2. Mechanistic studies showed that BBR treatment induced an enhancement of miR-24-3p. PIM-2 is a direct target of miR-24-3p. Blockade of PIM-2 or miR-24-3p reversed BBR-induced cell apoptosis. In vivo studies, BBR remarkably alleviated leukemia conditions in a EU4 xenograft mouse model, whereas inhibition of miR-24-3p significantly reversed the effects of BBR in the leukemia condition. Conclusions: miR-24-3p/PIM-2/XIAP signaling contributes to BBR-mediated leukemia mitigation in p53-defect ALL, which should be further developed as a treatment strategy in ALL patients with p53 deficiency. Methods: Cell viability and apoptosis were determined using CCK-8 and TUNEL assays, respectively. The dual-luciferase reporter gene system was used to determine the interaction between miR-24-3p and 3′-untranslated regions (UTRs) of PIM-2.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, whereas it is less common in adults. Identification of cytogenetic aberrations and a small number of molecular abnormalities are still the most important risk and therapy stratification methods in clinical practice today. Next generation sequencing (NGS) technology provides a large amount of data contributing to elucidation of mutational landscape of childhood (cALL) and adult ALL (aALL).We analyzed DNA samples from 34 cALL and aALL patients, using NGS targeted sequencing TruSeq Amplicon - Cancer Panel (TSACP) which targets mutational hotspots in 48 cancer related genes.We identified a total of 330 variants in the coding regions, out of which only 95 were potentially protein-changing. Observed in individual patients, detected mutations predominantly disrupted Ras/RTK pathway (STK11, KIT, MET, NRAS, KRAS, PTEN). Additionally, we identified 5 patients with the same mutation in HNF1A gene, disrupting both Wnt and Notch signaling pathway. In two patients we detected variants in NOTCH1 gene. HNF1A and NOTCH1 variants were mutually exclusive, while genes involved in Ras/RTK pathway exhibit a tendency of mutation accumulation.Our results showed that ALL contains low number of mutations, without significant differences between cALL and aALL (median per patient 2 and 3, respectively). Detected mutations affect few key signaling pathways, primarily Ras/RTK cascade. This study contributes to knowledge of ALL mutational landscape, leading to better understanding of molecular basis of this disease.Akutna limfoblastna leukemija (ALL) je najčešće maligno oboljenje kod dece, dok je kod odraslih njena učestalost mnogo niža. U današnjoj kliničkoj praksi kao najvažnije metode stratifikacije pacijenata u određene grupe rizika koriste se metode identifikacije citogenetičkih aberacija i malog broja molekulanih markera. Tehnologija sekvenciranja nove generacije (SNG) obezbeđuje veliku količinu podataka koji doprinose razjašnjavanju mutacionog profila dečje (dALL) i adultne ALL (aALL).Uzorci DNK iz 34 dALL i aALL pacijenata analizirani su primenom SNG ciljanog sekvenciranja ("TruSeq Amplicon Cancer Panel - TSACP") kojim se sekvenciraju "hotspot" mutacije u 48 gena povezanih sa kancerom.Identifikovano je ukupno 330 varijanti u kodirajućim regionima, od kojih je samo 95 njih za posledicu imalo potencijalnu promenu u proteinu. Posmatrano kod pojedinačnih pacijenata, detektovane mutacije su pretežno remetile Ras/RTK signalni put (STK11, KIT, MET, NRAS, KRAS, PTEN). Pored toga, identifikovano je 5 pacijenata sa istom mutacijom u HNF1A genu, koja je uzrokovala poremećaje u Wnt i Notch signalnom putu. Kod dva pa cijenta otkrivene su varijante u NOTCH1 genu. Nije detektovano istovremeno prisustvo varijanti u HNF1A i NOTCH1 genu, dok su geni uključeni u Ras/RTK signalni put pokazali tendenciju ka akumuliranju mutacija.Naši rezultati pokazuju da ALL sadrži Mali broj mutacija, bez značajnih razlika između dALL i aALL (medijana po pacijentu 2 odnosno 3). Detektovane mutacije izazivaju poremećaje u nekoliko ključnih signalnih puteva, prvenstveno Ras/RTK kaskade. Ova studija doprinosi ukupnom znanju o mutacionom profilu ALL, što vodi ka boljem razumijevanju molekularne osnove ovog oboljenja.

Background Acute lymphoblastic leukemia is the most common type of pediatric cancer. Acute lymphoblastic leukemia causes an altered bone mineral homeostasis state, which can contribute to osteopenia, and bone fractures, most commonly vertebral fractures. With the increasing number of childhood cancer survivors, late adverse effects such as musculoskeletal comorbidities are often reported and are further influenced by inactive lifestyle habits. Physical activity has been shown to increase the mechanical workload of the bone, mitigating bone impairment in other cancer-specific populations. Objective This interventional pilot study aims to investigate the use of telehealth to deliver a home-based exercise intervention for early-on survivors of bone marrow–related hematological malignancies and to assess its impact on survivors’ musculoskeletal and functional health. Methods We aimed to recruit a group of 12 early-on survivors of acute lymphoblastic leukemia, within 6 months to 5 years of treatment, to participate in and complete the proposed telehealth intervention with a parent. The 16-week intervention included 40 potential home-based physical activity interventions supervised by a kinesiologist through a telehealth internet platform, with monthly progression. Patients were recruited to the cohort if they were able to participate in the intervention during the first month (minimum 12 weeks of intervention). Evaluation before and after the intervention protocol highlighted differences in functional capacities and musculoskeletal health of patients using mechanography, peripheral quantitative computed tomography, 6-minute walk test, and grip force test. Results The recruitment rate for the intervention was low (12/57, 21% of contacted patients). Of 12 patients, 3 were excluded (1=relapse, 1=failure to meet technical requirements, and 1=abandoned). The 9 patients who completed the intervention (6 girls; mean age 10.93, SD 2.83 years; mean BMI 21.58, SD 6.55 kg/m2; mean time since treatment completion 36.67, SD 16.37 months) had a mean adherence of 89% and a completion rate of 75%. In addition, these patients showed functional improvements in lower limb muscle force and power as well as in the 6-minute walk test distance. Participants also showed improved bone health after the intervention on the following parameters: bone mineral content, stress-strain index, total and cortical cross-sectional area at the 14% site (P=.03, P=.01, P=.01, and P=.001, respectively) and 38% site of the tibia (P=.003, P=.04, P=.001, and P=.003, respectively). Conclusions High adherence and participation rates suggest that telehealth is a feasible method to deliver exercise interventions to young early-on survivors of acute lymphoblastic leukemia. The proposed intervention seems promising in providing benefits to patients’ functional performance and bone health, but a large-scale study is needed to confirm this assumption.

The recombination mediated by recombination activating gene (RAG) is not only the dominant mutational process but also the predominant driver of oncogenic genomic rearrangement in acute lymphoblastic leukemia (ALL). It is further responsible for leukemic clonal evolution. In this study, significant RAG1 increase is observed in the subsets of B-ALL patients, and high expression of RAG1 is observed to be correlated with high proliferation markers. IKZF1-encoded protein, IKAROS, directly binds to the RAG1 promoter and regulates RAG1 expression in leukemic cells. CK2 inhibitor by increasing IKAROS activity significantly suppresses RAG1 expression in ALL in an IKAROS-dependent manner. Patients with IKZF1 deletion have significantly higher expression of RAG1 compared to that without IKZF1 deletion. CK2 inhibitor treatment also results in an increase in IKZF1 binding to the RAG1 promoter and suppression of RAG1 expression in primary ALL cells. Taken together, these results demonstrate that RAG1 high expression is associated with high proliferation markers in B-ALL. Our data for the first time proved that RAG1 expression is directly suppressed by IKAROS. Our results also reveal drive oncogenesis of B-ALL is driven by high expression of RAG1 with IKAROS dysfunction together, which have significance in an integrated prognostic model for adult ALL.

Acute lymphoblastic leukemia (ALL) is a common malignant hematologic disease that is characterized by large numbers of dedifferentiated lymphoid cells. Statistical data of ALL's incidence and mortality are fundamental for policymakers to allocate resources optimally. In this study, we reported the incidence, death, and disability-adjusted life year (DALY) of ALL in the globe from 1990 to 2017. Our analysis showed that the incidence case of ALL increased by 30.81%, while the age-standardized incidence rate (ASIR) maintained stable. Subgroup analysis by social-demographic index (SDI) showed that ALL's ASIR was significantly decreased in high SDI countries, but were moderately increased in high-middle SDI countries. The change trends of age-standardized death rate and DALY rate were similar to ASIR trends. Subgroup analysis by age groups showed that children and the elderly were more likely to suffer ALL. Risk factor analysis demonstrated that smoking was the most significant contributor to ALL's death and DALY in the globe. Besides, the high body-mass index is playing an increasingly important role in ALL-caused mortality. Multiple methods to counteract potential risk factors should be adopted, such as controlling body-mass index in all regions and avoiding occupational exposure to carcinogens in low SDI countries.

Introduction Acute lymphoblastic leukemia (ALL) is one of the most common cancers in children and accounts for about ⅓ of cancers in children. The annual incidence of ALL is 4 patients per 100,000 children. Their peak age is between 2-5 Year. One of the most important prognostic factors is cytogenetic abnormalities which are very effective in determining treatment policy. Aim To determine the frequency of cytogenetic findings and its effect on the outcome of children with ALL. Materials and methods This retrospective cross-sectional analytical study was conducted on children with ALL who their disease was diagnosed between 2001and 2009. Furthermore, 206 patients with ALL were examined by referring to Clinic of Ali Asghar Hospital in Tehran. Data was collected from medical records and analyzed by SPSS16 software. Results 206 children with ALL were enrolled in the study. The estimated event-free survival rate of all enrolled patients was more than 70%. There was a significant relationship between type of cytogenetic disorder and clinical outcome of patients (P˂0.0001), where the highest mortality was observed in patients with t (9;22) and t (4;11). There was no significant correlation between the sex and age with the clinical outcome of the patient (P = 0.064; p=0.322). There was a statistically significant relationship between mediastinal mass and clinical outcome (P = 0.002), indicating that the presence of cells growth in an involuntary way can be cause of the cancer. A significant association was found between the clinical outcome of patients and radiotherapy (P = 0.043), indicating that radiotherapy is effective in improving cancer. Conclusion The findings demonstrated that the average survival rate without recurrence in children was at level of the European countries. However, the strong chemotherapy weakened the role of many prognostic factors in ALL patients, but some translocations are prognostic factors in predicting death in patients with ALL. Therefore, patients with this factor need to receive more confident treatment policy. Comprehensive studies are required by focusing on more samples because of low number of relapses and deaths in the present study.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Numerous studies have shown that microRNAs (miRNAs) could play a role in this disease. Nowadays, more than 2500 miRNAs have been described, that regulate more than 50% of genes, including those involved in B-cell maturation, differentiation and proliferation. Genetic variants in miRNAs can alter their own levels or function, affecting their target gene expression, and then, may affect ALL risk. Therefore, the aim of this study was to determine the role of miRNA genetic variants in B-ALL susceptibility. We analyzed all variants in pre-miRNAs (MAF > 1%) in two independent cohorts from Spain and Slovenia and inferred their functional effect by in silico analysis. SNPs rs12402181 in miR-3117 and rs62571442 in miR-3689d2 were associated with ALL risk in both cohorts, possibly through their effect on MAPK signalling pathway. These SNPs could be novel markers for ALL susceptibility.

Hematopoietic stem cell transplantation (HSCT) is being increasingly explored as a treatment modality for older patients with acute lymphoblastic leukemia (ALL). Yet, concerns regarding the long term outcome of transplantation in older patients limit the wide spread applicability of this approach. In this analysis we set out to determine the outcome of ALL patients over the age of 60 who underwent reduced intensity HSCT. Herein, we present the experience of the acute leukemia working party (ALWP) of the EBMT in this age group. We analyzed a cohort of 142 patients transplanted in first remission with a median age of 62 (range 60–76 years) and a median follow-up period of 36 months post-transplant. At 3 years, overall survival (OS) and leukemia-free survival were 42% and 35%, respectively. Multivariate analyses identified cytomegalovirus (CMV) donor-recipient matching (CMV D+/R+) to be significantly associated with inferior OS. Patients transplanted from unrelated donors experienced increased grade II-IV acute graft versus host disease compared to those receiving grafts from matched related donors [Hazard ratio (HR) of 3.7, 95% confidence interval (CI), 1.75–7.8; p = 0.0005). Outcome was not impacted by Philadelphia chromosome status. A select subset of older ALL patients will benefit from extended survival and a disease free state following HSCT.

// Qianqian Hou 1, * , Fei Liao 1, * , Shouyue Zhang 1, * , Duyu Zhang 1, * , Yan Zhang 2 , Xueyan Zhou 1 , Xuyang Xia 1 , Yuanxin Ye 8 , Hanshuo Yang 3 , Zhaozhi Li 1 , Leiming Wang 4 , Xi Wang 5 , Zhigui Ma 6 , Yiping Zhu 6 , Liang Ouyang 3 , Yuelan Wang 1 , Hui Zhang 7 , Li Yang 3 , Heng Xu 1, 8 , Yang Shu 1 1 Department of Laboratory Medicine, Precision Medicine Center, State Key Laboratory of Biotherapy and Precision Medicine Key Laboratory of Sichuan Province, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, Sichuan, China 2 Department of Thoracic Oncology, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China 3 State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, Sichuan, China 4 Department of Molecular Biology, Baylor College of Medicine, Houston, Texas, USA 5 Department of Microbiology, Immunology and Molecular Genetics, University of California at Los Angles, Los Angles, California, USA 6 Department of Pediatric Hematology/Oncology, West China Second Hospital, Sichuan University, Chengdu, Sichuan, China 7 Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, TN, USA 8 Department of Laboratory Medicine, Research Center of Clinical Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, China * These authors contributed equally to this work Correspondence to: Heng Xu, email: xuheng81916@scu.edu.cn Yang Shu, email: shuyang1986@gmail.com Keywords: GATA3, acute lymphoblastic leukemia, tissue-specific regulation network, microarray datasets Received: February 21, 2017 Accepted: March 11, 2017 Published: March 21, 2017 ABSTRACT GATA3 polymorphisms were reported to be significantly associated with susceptibility of pediatric B-lineage acute lymphoblastic leukemia (ALL), by impacting on GATA3 expression. We noticed that ALL-related GATA3 polymorphism located around in the tissue-specific enhancer, and significantly associated with GATA3 expression. Although the regulatory network of GATA3 has been well reported in T cells, the functional status of GATA3 is poorly understood in B-ALL. We thus conducted genome-wide gene expression association analyses to reveal expression associated genes and pathways in nine independent B-ALL patient cohorts. In B-ALL patients, 173 candidates were identified to be significantly associated with GATA3 expression, including some reported GATA3 -related genes (e.g., ITM2A ) and well-known tumor-related genes (e.g., STAT4 ). Some of the candidates exhibit tissue-specific and subtype-specific association with GATA3 . Through overexpression and down-regulation of GATA3 in leukemia cell lines, several reported and novel GATA3 regulated genes were validated. Moreover, association of GATA3 expression and its targets can be impacted by SNPs (e.g., rs4894953), which locate in the potential GATA3 binding motif. Our findings suggest that GATA3 may be involved in multiple tumor-related pathways (e.g., STAT/JAK pathway) in B-ALL to impact leukemogenesis through epigenetic regulation.

Relapse is the major cause of treatment-failure in adults with B-cell acute lymphoblastic leukemia (ALL) achieving complete remission after induction chemotherapy. Greater precision identifying persons likely to relapse is important. We did bio-informatics analyses of transcriptomic data to identify mRNA transcripts aberrantly-expressed in B-cell ALL. We selected 9 candidate genes for validation 7 of which proved significantly-associated with B-cell ALL. We next focused on function and clinical correlations of the cysteine and glycine-rich protein 2 (CSRP2). Quantitative real-time polymerase chain reaction (RT-qPCR) was used to examine gene transcript levels in bone marrow samples from 236 adults with B-cell ALL compared with samples from normals. CSRP2 was over-expressed in 228 out of 236 adults (97%) with newly-diagnosed B-cell ALL. A prognostic value was assessed in 168 subjects. In subjects with normal cytogenetics those with high CSRP2 transcript levels had a higher 5-year cumulative incidence of relapse (CIR) and worse relapse-free survival (RFS) compared with subjects with low transcript levels (56% [95% confidence interval, 53, 59%] vs. 19% [18, 20%]; P = 0.011 and 41% [17, 65%] vs. 80% [66-95%]; P = 0.007). In multivariate analyses a high CSRP2 transcript level was independently-associated with CIR (HR = 5.32 [1.64-17.28]; P = 0.005) and RFS (HR = 5.56 [1.87, 16.53]; P = 0.002). Functional analyses indicated CSRP2 promoted cell proliferation, cell-cycle progression, in vitro colony formation and cell migration ability. Abnormal CSRP2 expression was associated with resistance to chemotherapy; sensitivity was restored by down-regulating CSRP2 expression.

The survival rate of childhood acute lymphoblastic leukemia (ALL) is approaching 90%, while the prognosis of adults remains poor due to the limited therapeutic approaches. In order to identify new targets for ALL, we performed whole-exome sequencing on four adults with B-ALL and discovered a somatic JAK1 S646P mutation. Sanger sequencing of JAK1 was conducted on 53 ALL patients, and two cases exhibited A639G and P960S mutations separately. Functional studies demonstrated that only JAK1 S646P mutation could activate multiple signaling pathways, drive cytokine-independent cell growth, and promote proliferation of malignant cells in nude mice. Moreover, a high sensitivity to the JAK1/2 inhibitor ruxolitinib was observed in S646P mutant model. Exploration in a total of 209 ALL cases showed that JAK1 mutations occur at a frequency of 10.5% in T-ALL (2/19) and 1.6% in B-ALL (3/190). Collectively, our results suggested that JAK1 S646P is an activating mutation in vitro and in vivo. JAK-STAT pathway might represent a promising therapeutic target for ALL.

// Zheng Ge 1, 2 , Xilian Zhou 3 , Yan Gu 3 , Qi Han 3 , Jianyong Li 3 , Baoan Chen 1, 2 , Qinyu Ge 4 , Elanora Dovat 5 , Jonathon L. Payne 5, 6 , Tianyu Sun 7 , Chunhua Song 2, 5 , Sinisa Dovat 2, 5 1 Department of Hematology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009, China 2 International Cooperative Leukemia Group and International Cooperative Laboratory of Hematology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009, China 3 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing 210029, China 4 State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China 5 Department of Pediatrics, Pennsylvania State University Medical College, Hershey, PA 17033, USA 6 Loma Linda University School of Medicine, Department of Basic Sciences, Loma Linda, CA 92350, USA 7 Department of Internal Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA 19140, USA Correspondence to: Zheng Ge, email: Janege879@hotmail.com Sinisa Dovat, email: sdovat@hmc.psu.edu Keywords: BCL6, BACH2, IKZF1, acute lymphoblastic leukemia Received: October 11, 2016 Accepted: November 24, 2016 Published: December 20, 2016 ABSTRACT B-Cell CLL/Lymphoma 6 ( BCL6 ) is a proto-oncogene that is highly expressed in acute lymphoblastic leukemia (ALL). BTB and CNC Homology 1 Basic Leucine Zipper Transcription Factor 2 (BACH2) is a suppressor of transcription. The BACH2 – BCL6 balance controls selection at the pre-B cell receptor checkpoint by regulating p53 expression. However, the underlying mechanism and the clinical relevance of the BCL6/BACH2 axis are unknown. Here, we found that Ikaros, a tumor suppressor encoded by IKZF1 , directly binds to both the BCL6 and BACH2 promoters where it suppresses BCL6 and promotes BACH2 expression in B-cell ALL (B-ALL) cells. Casein kinase 2 (CK2) inhibitors increase Ikaros function thereby inhibiting BCL6 and promoting BACH2 expression in an Ikaros-dependent manner. We also found that the expression of BCL6 is higher while BACH2 expression is lower in patients with B-ALL than normal bone marrow control. High BCL6 and low BACH2 expression is associated with high leukemic cell proliferation, unfavorable clinical and laboratory features, and inferior outcomes. Moreover, IKZF1 deletion is associated with high BCL6 and low BACH2 expression in B-ALL patients. CK2 inhibitors increase Ikaros binding to the promoter of BCL6 and BACH2 and suppress BCL6 while promoting BACH2 expression in the primary B-ALL cells. Our data indicates that Ikaros regulates expression of the BCL6/BACH2 axis in B-ALL. High BCL6 and low BACH2 expression are associated with Ikaros dysregulation and have a potential effect on the development of B-ALL.

Acute lymphoblastic leukemia is the most common type of childhood cancer, accounting for about 23% of all cancers diagnosed in this age group. The last stage of radical treatment is remission maintenance, during which hospitalization is not necessary. The lesions occurring in the oral cavity caused by medications and chemotherapy may also be directly related to hematological and systemic disorders. The aim of this study was to examine the levels of selected pro- and anti-inflammatory cytokines in saliva and serum of both patients undergoing remission maintenance and those after the cessation of therapy who reported to the hematology clinic of the Pediatric University Hospital in Lublin. The results were later analyzed in relation to the frequency of oral lesions and subjective intensity of oral complaints. The study revealed significant differences in salivary and serum concentrations of TNF- and IL-10 between test and control groups. Oral lesions were more frequent in patients receiving therapy compared to the control group. Subjective afflictions described by the Visual Analog Scale (VAS) mean values were highest in the control group.

Background Suboptimal adherence to 6-mercaptopurine (6-MP) is prevalent in pediatric acute lymphoblastic leukemia (ALL) and associated with increased risk of relapse. Rapid uptake of personal technology makes mobile health (mHealth) an attractive platform to promote adherence. Objective Study objectives were to examine access to mobile technology and preferences for an mHealth intervention to improve medication adherence in pediatric ALL. Methods A cross-sectional survey was administered in oncology clinic to parents of children with ALL as well as adolescents and young adults (AYAs) with ALL receiving maintenance chemotherapy. Results A total of 49 parents (median age [IQR] 39 [33-42] years; female 76% [37/49]) and 15 patients (median age [IQR] 17 [16-19]; male 80% [12/15]) participated. All parents and AYAs owned electronic tablets, smartphones, or both. Parents’ most endorsed mHealth app features included a list of medications (71%, 35/49), information about 6-MP (71%, 35/49), refill reminders (71%, 35/49), and reminders to take 6-MP (71%, 35/49). AYAs' most endorsed features included refill reminders (73%, 11/15), reminders to take 6-MP (73%, 11/15), and tracking 6-MP (73%, 11/15). Conclusions Parents and AYAs reported ubiquitous access to mobile technology and strong interest in multiple adherence-specific mHealth app features. Parents and AYAs provided valuable insight into preferred features for a multifunctional behavioral intervention (mHealth app) to promote medication adherence in pediatric ALL.

// Haijun Zhao 1, 5, * , Pengcheng Shi 1, * , Manman Deng 2, * , Zhiwu Jiang 3 , Yin Li 1 , Vinodh Kannappan 4 , Weiguang Wang 4 , Peng Li 3 , Bing Xu 1, 2 1 Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou, P. R. China 2 Department of Hematology, The First Affiliated Hospital of Xiamen University, Xiamen, P. R. China 3 Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China 4 Research Institute in Healthcare Science, Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK 5 Department of Hematology, Anqing Municipal Hospital of Anhui Medical University, Anqing, P. R. China * These authors have contributed equally to this work Correspondence to: Bing Xu, email: xubingzhangjian@126.com Peng Li, email: li_peng@gibh.ac.cn Weiguang Wang, email: w.wang2@wlv.ac.uk Keywords: triptolide, chemotherapy, drug resistance, DNA damage, acute lymphoblastic leukemia Received: June 30, 2016 Accepted: October 19, 2016 Published: November 19, 2016 ABSTRACT Chemoresistance represents a major challenge for treatment of acute lymphoblastic leukemia (ALL). Thus, new drugs to overcome chemoresistance in ALL are urgently needed. To this end, we established a cytarabine (araC)-resistant ALL cell line (NALM-6/R), which interestingly displayed cross-resistance towards doxorubicin (ADM). Here we report that low dose of triptolide (TPL), a natural product used for treating inflammatory diseases such as arthritis, could reverse araC and ADM resistance and in NALM-6/R cells as well as primary cells from patients with relapsed or refractory (R/R) ALL, reflected by inhibition of cell proliferation and induction of apoptosis in vitro , and repression of tumor growth in vivo in a mouse xenograft model. Mechanistically, these events were associated with impaired mitochondrial membrane potential and increased reactive oxygen species (ROS) production. Co-treatment with TPL and araC or ADM upregulated pro-apoptotic caspase-9 protein, inhibited checkpoint kinase 1 (Chk1) and 2 (Chk2) phosphorylation, and induced γH2A.X (a DNA damage marker). Notably, the combination regimen of TPL and conventional chemotherapeutics also rapidly diminished tumor burden in a patient with R/R ALL. Together, these findings provide preclinical evidence for repurposing use of TPL in combination with chemotherapeutic agents to treat R/R ALL as an alternative salvage regimen.

Unlike chronic myeloid leukemia, patients with acute lymphoblastic leukemia (ALL) with Philadelphia chromosome (Ph+) do not respond well to Imatinib or tyrosine kinase inhibitors (TKI). In addition, TKI might induce resistant mutations in kinase domain (KD) of ABL in patients with relapsed diseases. Of the histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) has shown to induce potent cytotoxicity on acute myeloid leukemia cell lines but Givinostat effect on acute lymphoblastic leukemia (ALL) has not been reported. We investigated if Givinostat could exert similar inhibitory effect on SUP-B15, an established B-cell ALL with Philadelphia chromosome (Ph+). Two Ph+ leukemia cell lines, SUP-B15 and an AML cell line K562 were studied in parallel for their responses to Givinostat. Mutation status of TP53 genes was also examined to correlate cellular proliferation and apoptosis. Givinostat significantly inhibited cell proliferation of SUP-B15 (IC50:0.18±0.03μM) and simultaneously inhibited BCR-ABL signal pathway. A remarkable apoptosis was induced by 0.25μM Givinostat in SUP-B15 along with the activation of caspase cascades and increased expression of p21. These inhibitory and proapoptotic effects were not observed in K562 simultaneously treated with Givinostat. Finally our studies showed that TP53 mutation status might determine responder or non-responder to Givinostat in these two Ph+ leukemia cell lines.

To characterize the mutational patterns of acute lymphoblastic leukemia (ALL) we performed deep next generation sequencing of 872 cancer genes in 172 diagnostic and 24 relapse samples from 172 pediatric ALL patients. We found an overall greater mutational burden and more driver mutations in T-cell ALL (T-ALL) patients compared to B-cell precursor ALL (BCP-ALL) patients. In addition, the majority of the mutations in T-ALL had occurred in the original leukemic clone, while most of the mutations in BCP-ALL were subclonal. BCP-ALL patients carrying any of the recurrent translocations ETV6-RUNX1, BCR-ABL or TCF3-PBX1 harbored few mutations in driver genes compared to other BCP-ALL patients. Specifically in BCP-ALL, we identified ATRX as a novel putative driver gene and uncovered an association between somatic mutations in the Notch signaling pathway at ALL diagnosis and increased risk of relapse. Furthermore, we identified EP300, ARID1A and SH2B3 as relapse-associated genes. The genes highlighted in our study were frequently involved in epigenetic regulation, associated with germline susceptibility to ALL, and present in minor subclones at diagnosis that became dominant at relapse. We observed a high degree of clonal heterogeneity and evolution between diagnosis and relapse in both BCP-ALL and T-ALL, which could have implications for the treatment efficiency.

Relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem and is thought to be associated with clonal selection during treatment. In this study we used an established pre-clinical model of induction therapy to increase our understanding of the effect of engraftment and chemotherapy on clonal selection and acquisition of drug resistance in vivo. Immune-deficient mice were engrafted with patient diagnostic specimens and exposed to a repeated combination therapy consisting of vincristine, dexamethasone, L-asparaginase and daunorubicin. Any re-emergence of disease following therapy was shown to be associated with resistance to dexamethasone, no resistance was observed to the other three drugs. Immunoglobulin/T-cell receptor gene rearrangements closely matched those in respective diagnosis and relapse patient specimens, highlighting that these clonal markers do not fully reflect the biological changes associated with drug resistance. Gene expression profiling revealed the significant underlying heterogeneity of dexamethasone-resistant xenografts. Alterations were observed in a large number of biological pathways, yet no dominant signature was common to all lines. These findings indicate that the biological changes associated with T-ALL relapse and resistance are stochastic and highly individual, and underline the importance of using sophisticated molecular techniques or single cell analyses in developing personalized approaches to therapy.

// Xiaojing Lin 1, 5 , Xingli Zou 5 , Ziming Wang 2, 3 , Qin Fang 4 , Shuya Chen 1, 2, 3 , Jun Huang 1, 2, 3 , Nana Zhe 1, 2, 3 , Meisheng Yu 1, 2, 3 , Yaming Zhang 2, 3 , Jishi Wang 1, 2, 3 1 Clinical Medicine, Guizhou Medical University, Guiyang 550004, China 2 Department of Hematology, The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China 3 Department of Hematology, Guizhou Provincial Laboratory of Hematopoietic Stem Cell Transplantation Center, Guiyang 550004, China 4 Department of Pharmacy, The Affiliated Baiyun Hospital of Guizhou Medical University, Guiyang 550004, China 5 Department of Hematology, The Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China Correspondence to: Jishi Wang, email: wangjishi9646@163.com Keywords: acute lymphoblastic leukemia, BCR-ABL1-positive, heme oxygenase-1, IKZF1, STAT5 Received: September 16, 2015 Accepted: June 07, 2016 Published: July 20, 2016 ABSTRACT The correlation between Heme oxygenase-1 (HO-1) and dominant-negative Ikaros isoform 6 (IK6) is unclear. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL) by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. IK6-positive patients had an unfavorable outcome compared with IK6-negative ones. Further study showed that the level of HO-1 expression was higher in IK6-positive patients’ samples than that in IK6-negative ones. And there was a strong correlation between the expression of IK6 and HO-1. The growth of primary CD34 + leukemic cells derived from our IK6-positive patients’ pool was prohibited by silencing HO-1, further promoting their apoptosis. Furthermore, primary CD34 + leukemic cells derived from IK6-positive patients shown poor responses to imatinib in comparison with wild-type (IK1) patients, suggesting that the expression of IK6 resisted to imatinib in adult BCR-ABL1-positive B-ALL. Importantly, inhibition of HO-1 also increased their sensitivity to tyrosine kinase inhibitors (TKIs). Finally, we found that IK6 activated downstream STAT5, and HO-1 was one of the downstream target genes of STAT5. In conclusion, HO-1 is an essential survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can attenuate the negative impact of IK6.

// Zheng Ge 1 , Yan Gu 2 , Gang Zhao 1 , Jianyong Li 2 , Baoan Chen 1 , Qi Han 2 , Xing Guo 2 , Juan Liu 2 , Hui Li 3 , Michael D. Yu 4 , Justin Olson 5 , Sadie Steffens 3 , Kimberly J. Payne 6 , Chunhua Song 3 , Sinisa Dovat 3 1 Department of Hematology, Zhongda Hospital, Medical School of Southeast University, Nanjing, 210009, China 2 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, 210029, China 3 Department of Pediatrics, Pennsylvania State University Medical College, Hershey, PA, 17033, USA 4 Sydney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, 19107 USA 5 University of Wisconsin at Stout, Menomonie, WI, 54751, USA 6 Loma Linda University, Department of Pathology and Human Anatomy, Loma Linda, CA, 92350, USA Correspondence to: Zheng Ge, email: Janege879@hotmail.com Chunhua Song, email: csong@hmc.psu.edu Sinisa Dovat, email: sdovat@hmc.psu.edu Keywords: CRLF2, IKZF1, adult, acute lymphoblastic leukemia Received: June 06, 2016 Accepted: June 24, 2016 Published: July 06, 2016 ABSTRACT Overexpression of cytokine receptor-like factor 2 ( CRLF2 ) due to chromosomal rearrangement has been observed in acute lymphoblastic leukemia (ALL) and reported to contribute to oncogenesis and unfavorable outcome in ALL. We studied B-ALL and T-ALL patients without CRLF2 rearrangement and observed that CRLF2 is significantly increased in a subset of these patients. Our study shows that high CRLF2 expression correlates with high-risk ALL markers, as well as poor survival. We found that the IKZF1 -encoded protein, Ikaros, directly binds to the CRLF2 promoter and regulates CRLF2 expression in leukemia cells. CK2 inhibitor, which can increase Ikaros activity, significantly increases Ikaros binding in ALL cells and suppresses CRLF2 expression in an Ikaros-dependent manner. CRLF2 expression is significantly higher in patients with IKZF1 deletion as compared to patients without IKZF1 deletion. Treatment with CK2 inhibitor also results in an increase in IKZF1 binding to the CRLF2 promoter and suppression of CRLF2 expression in primary ALL cells. We further observed that CK2 inhibitor induces increased H3K9me 3 histone modifications in the CRLF2 promoter in ALL cell lines and primary cells. Taken together, our results demonstrate that high expression of CRLF2 correlates with high-risk ALL and short survival in patients without CRLF2 rearrangement. Our results are the first to demonstrate that the IKZF1 -encoded Ikaros protein directly suppresses CRLF2 expression through enrichment of H3K9me 3 in its promoter region. Our data also suggest that high CRLF2 expression works with the IKZF1 deletion to drive oncogenesis of ALL and has significance in an integrated prognostic model for adult high-risk ALL.

// Zheng Ge 1, 2, 3 , Yan Gu 2 , Lichan Xiao 2 , Qi Han 2 , Jianyong Li 2 , Baoan Chen 1 , James Yu 4 , Yuka Imamura Kawasawa 5 , Kimberly J. Payne 6 , Sinisa Dovat 3 , Chunhua Song 3 1 Department of Hematology, Zhongda Hospital, Southeast University Medical School, Nanjing 210009, China 2 Department of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing 210029, China 3 Department of Pediatrics, Pennsylvania State University Medical College, Hershey, PA 17033, USA 4 Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA 5 Penn State Hershey Genome Sciences Facility, Penn State College of Medicine, Hershey, PA 17033, USA 6 Department of Pathology and Human Anatomy, Loma Linda University, Loma Linda, CA 92350, USA Correspondence to: Zheng Ge, email: Janege879@hotmail.com Sinisa Dovat, email: sdovat@hmc.psu.edu Chunhua Song, email: csong@hmc.psu.edu Keywords: IL7R , SH2B3 , Ikaros , gene expression, acute lymphoblastic leukemia Received: May 18, 2016 Accepted: June 03, 2016 Published: June 14, 2016 ABSTRACT Acute lymphoblastic leukemia (ALL) remains the leading cause of cancer-related death in children and young adults. Compared to ALL in children, adult ALL has a much lower cure rate. Therefore, it is important to understand the molecular mechanisms underlying high-risk ALL and to develop therapeutic strategies that specifically target genes or pathways in ALL. Here, we explored the IL7R and SH2B3 expression in adult ALL and found that IL7R is significantly higher and Sh2B3 lower expressed in B-ALL compared to normal bone marrow control, and the IL7R high SH2B3 low is associated with high-risk factors, and with high relapse rate and low disease-free survival rate in the patients. We also found that Ikaros deletion was associated with the IL7R high SH2B3 low expression pattern and Ikaros directly binds the IL7R and SH2B3 promoter, and suppresses IL7R and promotes SH2B3 expression. On the other hand, casein kinase inhibitor, which increases Ikaros function, inhibits IL7R and stimulates SH2B3 expression in an Ikaros dependent manner. Our data indicate that IL7R high SH2B3 low expression distinguishes a novel subset of high-risk B-ALL associated with Ikaros dysfunction, and also suggest the therapeutic potential for treatment that combines casein kinase inhibitor, as an Ikaros activator, with drugs that target the IL7R signaling pathway.

The bone marrow niche has a significant impact on acute lymphoblastic leukemia (ALL) cell phenotype. Of clinical relevance is the frequency with which quiescent leukemic cells, in this niche, survive treatment and contribute to relapse. This study suggests that marrow microenvironment regulation of BCL6 in ALL is one factor that may be involved in the transition between proliferative and quiescent states of ALL cells. Utilizing ALL cell lines, and primary patient tumor cells we observed that tumor cell BCL6 protein abundance is decreased in the presence of primary human bone marrow stromal cells (BMSC) and osteoblasts (HOB). Chemical inhibition, or shRNA knockdown, of BCL6 in ALL cells resulted in diminished ALL proliferation. As many chemotherapy regimens require tumor cell proliferation for optimal efficacy, we investigated the consequences of constitutive BCL6 expression in leukemic cells during co-culture with BMSC or HOB. Forced chronic expression of BCL6 during co-culture with BMSC or HOB sensitized the tumor to chemotherapy induced cell death. Combination treatment of caffeine, which increases BCL6 expression in ALL cells, with chemotherapy extended the event free survival of mice. These data suggest that BCL6 is one factor, modulated by microenvironment derived cues that may contribute to regulation of ALL therapeutic response.

In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL, n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%–18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring.

The epidermal growth factor receptor HER2/neu is expressed on various cancers and represents a negative prognostic marker, but is also a target for the therapeutic monoclonal antibody Trastuzumab. In about 30% of cases, HER2/neu is expressed on acute lymphoblastic leukemia (ALL) cells and was proposed to be associated with a deleterious prognosis. Here we evaluated clinical data from 65 ALL patients (HER2/neu+, n = 17; HER2/neu−, n = 48) with a median follow-up of 19.4 months (range 0.6-176.5 months) and observed no association of HER2/neu expression with response to chemotherapy, disease free or overall survival. In vitro, treatment of primary ALL cells (CD20+HER2/neu+, CD20+HER2/neu− and CD20−HER2/neu−) with Rituximab and Trastuzumab led to activation of NK cells in strict dependence of the expression of the respective antigen. NK reactivity was more pronounced with Rituximab as compared to Trastuzumab, and combined application could lead to additive effects in cases where both antigens were expressed. Besides providing evidence that HER2/neu expression is no risk factor in ALL patients, our data demonstrates that HER2/neu can be a promising target for Trastuzumab therapy in the subset of ALL patients with the potential to improve disease outcome.

BackgroundAcute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Caring for children with ALL is challenging for parents. A mobile health (mHealth) supportive care intervention was developed to meet parents’ needs. ObjectiveThis study aims to evaluate the potential effectiveness of this mHealth supportive care intervention on emotional distress, social support, care burden, uncertainty in illness, quality of life, and knowledge. MethodsWe conducted a quasi-experimental pre- and postdesign study from June 2015 to January 2016. In total, 101 parents were enrolled in the study, with 50 in the observation group and 51 in the intervention group. Parents in the observation group received the standard health education and were observed for 3 months. Parents in the intervention group received the mHealth supportive care intervention, in addition to the standard health education. The intervention consisted of 2 parts—an Android smartphone app “Care Assistant (CA)” and a WeChat Official Account. The CA with 8 modules (Personal Information, Treatment Tracking, Family Care, Financial and Social Assistance, Knowledge Center, Self- Assessment Questionnaires, Interactive Platform, and Reminders) was the main intervention tool, whereas the WeChat Official Account was supplementary to update information and realize interaction between parents and health care providers. Data of parents’ social support, anxiety, depression, care burden, uncertainty in illness, quality of life, their existing knowledge of ALL and care, and knowledge need were collected before and after the 3-month study period in both groups. For the intervention group, parents’ experience of receiving the intervention was also collected through individual interviews. ResultsOverall, 43 parents in the observation group and 49 in the intervention group completed the study. Results found that the intervention reduced parents’ anxiety (Dint(Post-Pre)=−7.0 [SD 13.1], Dobs(Post-Pre)=−0.4 [SD 15.8], t90=−2.200, P=.03) and uncertainty in illness (Dint(Post-Pre)=−25.0 [SD 8.2], Dobs(Post-Pre)=−19.8 [SD 10.1], t90=−2.761, P=.01), improved parents’ social function (Dint(Post-Pre)=9.0 [SD 32.8], Dobs(Post-Pre)=−7.5 [SD 30.3], t90=2.494, P=.01), increased parents’ knowledge of ALL and care (Dint(Post-Pre)=28.4 [SD 12.4], Dobs(Post-Pre)=17.2 [SD 11.9], t90=4.407, P

International audience; Pax5 is the guardian of the B cell identity since it primes or enhances the expression of B cell specific genes and concomitantly represses the expression of B cell inappropriate genes. The tight regulation of Pax5 is therefore required for an efficient B cell differentiation. A defect in its dosage can translate into immunodeficiency or malignant disorders such as leukemia or lymphoma. Pax5 is expressed from two different promoters encoding two isoforms that only differ in the sequence of their first alternative exon. Very little is known regarding the role of the two isoforms during B cell differentiation and the regulation of their expression. Our work aims to characterize the mechanisms of regulation of the expression balance of these two isoforms and their implication in the B cell differentiation process using murine ex vivo analyses. We show that these two isoforms are differentially regulated but have equivalent function during early B cell differentiation and may have functional differences after B cell activation. The tight control of their expression may thus reflect a way to finely tune Pax5 dosage during B cell differentiation process.

// Setareh Safavi 1 , Linda Olsson 1, 2 , Andrea Biloglav 1 , Srinivas Veerla 3 , Molly Blendberg 1 , Johnbosco Tayebwa 1 , Mikael Behrendtz 4 , Anders Castor 5 , Markus Hansson 6 , Bertil Johansson 1, 2 , Kajsa Paulsson 1 1 Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden 2 Department of Clinical Genetics, University and Regional Laboratories, Region Skane, Lund, Sweden 3 Division of Oncology and Pathology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden 4 Department of Pediatrics, Linkoping University Hospital, Linkoping, Sweden 5 Department of Pediatrics, Skane University Hospital, Lund University, Lund, Sweden 6 Division of Hematology, Skane University Hospital, Lund University, Lund, Sweden Correspondence to: Setareh Safavi, e-mail: setareh.safavi@med.lu.se Kajsa Paulsson, e-mail: kajsa.paulsson@med.lu.se Keywords: acute lymphoblastic leukemia, hypodiploidy, next generation sequencing, chromosomal instability Received: August 26, 2015 Accepted: October 17, 2015 Published: October 30, 2015 ABSTRACT Purpose: To investigate the genetic and epigenetic landscape of hypodiploid (

Enhanced rRNA synthesis is a downstream effect of many of the signaling pathways that are aberrantly activated in cancer, such as the PI3K/mTOR and MAP kinase pathways. Recently, two new rRNA synthesis inhibitors have demonstrated therapeutic effects on cancer cells while sparing normal cells. One of them, CX-5461, is currently in phase 1 clinical trials for hematological malignancies. Here, we investigate the effectiveness of transient treatment with this drug on acute lymphoblastic leukemia cells. Our results show that short exposure to CX-5461 followed by drug washout is sufficient to induce persistent G2 cell-cycle arrest and irreversible commitment to cell death, in spite of rRNA synthesis returning to normal within 24 hours of drug washout. The magnitude of cell death after transient exposure is similar to continuous exposure, but the time to cell death is relatively delayed with transient exposure. In this report, we also investigate rational drug combinations that can potentiate the effect of continuous CX-5461 treatment. We show that the checkpoint abrogator UCN-01 can relieve CX-5461-induced G2 arrest and potentiate the cytotoxic effects of CX-5461. Finally, we show that ERK1/2 is activated upon CX-5461 treatment, and that pharmacological inhibition of MEK1/2 leads to enhanced cell death in combination with CX-5461. In summary, our results provide evidence for the effectiveness of CX-5461 pulse treatment, which may minimize drug related toxicity, and evidence for enhanced effectiveness of CX-5461 in combination with other targeted agents.

Although steady improvements to chemotherapeutic treatments has helped cure 80% of childhood acute lymphoblastic leukemia (ALL) cases, chemotherapy has proven to be less effective in treating the majority of adult patients, leaving allogeneic hematopoietic stem cell transplantation (allo-HSCT) as the primary adult treatment option. Nevertheless relapse are the leading cause of death following allo-HSCT. The genetic pathogenesis of relapse following allo-HSCT in Philadelphia chromosome- negative ALL (Ph- ALL) remains unexplored. We performed longitudinal whole-exome sequencing analysis in three adult patients with Ph- B-cell ALL (Ph- B-ALL) on samples collected from diagnosis to relapse after allo-HSCT. Based on these data, we performed target gene sequencing on 23 selected genes in 58 adult patients undergoing allo-HSCT with Ph- B-ALL. Our results revealed a significant enrichment of mutations in epigenetic regulators from relapsed samples, with recurrent somatic mutations in SETD2, CREBBP, KDM6A and NR3C1. The relapsed samples were also enriched in signaling factor mutations, including KRAS, PTPN21, MYC and USP54. Furthermore, we are the first to reveal the clonal evolution patterns during leukemia relapse after allo-HSCT. Cells present in relapsed specimens were genetically related to the diagnosed tumor, these cells therefore arose from either an existing subclone that was not eradicated by allo-HSCT therapy, or from the same progenitor that acquired new mutations. In some cases, however, it is possible that leukemia recurrence following allo-HSCT could result from a secondary malignancy with a distinct set of mutations. We identified novel genetic causes of leukemia relapse after allo-HSCT using the largest generated data set to date from adult patients with Ph- B-ALL.

Deletion of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) is well known in many hematologic malignancies, but only few reports have investigated this deletion effect on clinical prognosis. This study performed analysis of the CDKN2 deletion in 215 adult B- lineage acute lymphoblastic leukemia (B-ALL) patients, and related cytogenetic prognostic factors (BCR/ABL; E2A/PBXl; TEL/AML1; Mixed Lineage Leukemia (MLL) rearrangement; MYC, Immunoglobulin heavy locus (IGH) translocation). The prevalence of CDKN2 deletions in all study populations was 28.4%. There is no difference between patients with CDKN2 deletion and wild-type patients in sex, age, white blood cells (WBC) count, BM blast percentage, extra infiltration and induction complete remission (CR) rate. Analysis in relapse patients revealed that the distribution of CDKN2 deletion is higher in relapse patients (44.6%) than all patients (28.4%, P=0.006). Deletion of CDKN2 was significantly associated with poor outcomes including decreased overall survival (OS) (P

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

Ribosome biogenesis is a fundamental cellular process and is elevated in cancer cells. As one of the most energy consuming cellular processes, it is highly regulated by signaling pathways in response to changing cellular conditions. Many of the regulators of this process are aberrantly activated in various cancers. Recently two novel rRNA synthesis inhibitors, CX-5461 and BMH-21, have been shown to selectively kill cancer cells while sparing normal cells. Here, we tested the effectiveness of pre-rRNA synthesis inhibitor CX-5461 on acute lymphoblastic leukemia cells with different cytogenetic abnormalities. Acute lymphoblastic leukemia cells are more sensitive to rRNA synthesis inhibition compared to normal bone marrow cells. CX-5461 treated cells undergo caspase-dependent apoptosis independent of their p53 status. More-over, CX5461, activates checkpoint kinases and arrests cells in G2 phase of cell cycle. Finally, overcoming this G2 arrest by inhibiting ATR kinase leads to robust cell killing. These results show that CX-5461 can be even more potent in combination with ATR inhibitors.

Contains fulltext : 155199.pdf (Publisher’s version ) (Open Access) Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric leukemias. In this study, we determined CREB phosphorylation and mRNA levels showing that CREB expression was significantly higher in ALL compared to normal bone marrow (phosphorylation: P < 0.0001, mRNA: P = 0.004). High CREB and phospho-CREB expression was correlated with a lower median overall survival in a cohort of 140 adult ALL patients. ShRNA mediated knockdown of CREB in ALL cell lines blocked leukemic cell growth by inducing cell cycle arrest and apoptosis. Gene expression array analysis showed downregulation of CREB target genes regulating cell proliferation and glucose metabolism and upregulation of apoptosis inducing genes. Similar to CREB knockdown, the CREB inhibitor KG-501 decreased leukemic cell viability and induced apoptosis in ALL cell lines, as well as primary T-ALL samples, with cases showing high phospho-CREB levels being more sensitive than those with lower phospho-CREB levels. Together, these in vitro findings support an important role for CREB in the survival of ALL cells and identify this transcription factor as a potential target for treatment.