ALBERT

Description: Limited number of hematopoietic stem cells in umbilical cord blood (UCB) presents a problem when using UCB for stem cell transplantation. Improving their homing capacity could reduce the need for high initial cell numbers during transplantation procedures. Although it is evident that protein kinase B (PKB/c-Akt) plays an important role in regulation of migration of various cell types, a role for PKB in regulation of migration and homing of human hematopoietic stem and progenitor cells remains to be determined. PKB activity was found to be required for induction of adhesion to bone marrow–derived stromal cells and detrimental for migration of UCB-derived CD34+ hematopoietic progenitors. In addition, PKB activity was found to positively regulate integrin expression. CD34+ hematopoietic progenitors, and their capacity to form colonies in vitro, were not affected by transient inhibition of PKB. Finally, transplantation of β2-microglobulin−/− nonobese diabetic/severe combined immunodeficient mice with CD34+ cells ectopically expressing constitutively active PKB resulted in reduced migration to the bone marrow, whereas inhibition of PKB activity resulted in an induction in bone marrow homing and engraftment. These results indicate that transient inhibition of PKB activity may provide a means for ex vivo stem cell manipulation to improve bone marrow transplantation regimes.

Description: Background and Objectives Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognosis associated with ETV6-RUNX1 positive ALL, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. Leukemic cells reside in the bone marrow microenvironment, where they are nurtured and protected against chemotherapy. In this study, we investigated novel ways to disrupt this leukemic niche by targeting signaling pathways contributing to the migration of ETV6-RUNX1 positive leukemic cells. Results Gene expression profiling and subsequent pathway analysis of leukemic blasts of 654 ALL patients revealed a significant enrichment of genes involved in regulation of cellular movement and cell morphology in ETV6-RUNX1 positive BCP-ALL patients compared with ETV6-RUNX1 negative BCP-ALL patients (p 〈 0.001). In correspondence, the same pathways were significantly upregulated in cord blood-derived hematopoietic progenitor cells (CB-CD34+) ectopically expressing ETV6-RUNX1 (p 〈 10E-06). LARG (ARHGEF12) was identified to be the most important regulator of this pro-migratory signature. This gene encodes for the G-protein-regulated Rho Guanine Exchange Factor 12, a specific activator of the GTPase RhoA. LARG expression was 5.7-fold higher in ETV6-RUNX1 positive BCP-ALL cells than in ETV6-RUNX1 negative BCP-ALL cells (p 〈 10E-06). Similarly, LARG was upregulated 5.4-fold in CB-CD34+ cells expressing ETV6-RUNX1 compared with empty vector controls (p = 0.03). To determine the importance of the LARG/RhoA pathway in the induction of this migratory phenotype, we used two recently identified small molecule inhibitors of the LARG/RhoA pathway: Y16, a specific LARG inhibitor, and G04 (Rhosin), a specific RhoA inhibitor. Both inhibitors significantly reduced the migration of ETV6-RUNX1 positive leukemic cells towards a gradient of CXCL12 (75%, p 〈 0.01) whereas the migration potential of ETV6-RUNX1 negative leukemic cells remained unaffected. Migration of ETV6-RUNX1 positive leukemic cells towards patient derived mesenchymal stromal cells was inhibited to a similar extent (75-85%, p 〈 0.01). In contrast, LARG/RhoA inhibition in ETV6-RUNX1 negative cells resulted in an induction of migration towards MSCs. While LARG/RhoA inhibitors reduced migration of ETV6-RUNX1 positive cells in a targeted manner, inhibition of CXCR4 by the CXCR4 antagonist AMD3100 (Plerixafor) reduced the migration of both ETV6-RUNX1 positive cells and ETV6-RUNX1 negative BCP-ALL cells. Next, we studied the additional cellular effects of LARG/RhoA inhibition on leukemic cells. shRNA-mediated silencing of LARG modestly reduced the proliferation rate of both ETV6-RUNX1 positive and ETV6-RUNX1 negative cell lines (p

Description: Background: Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognostic parameters of this B-ALL subgroup, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. The molecular mechanisms underlying ETV6-RUNX1-driven leukemia are largely unknown. Increased knowledge of these mechanisms is essential to develop novel therapeutic strategies to selectively target ETV6-RUNX1-positive leukemia. Objectives: This study aims to identify and target the molecular drivers behind ETV6-RUNX1-positive BCP-ALL. Results: Gene expression profiling of leukemic blasts of 654 ALL patients revealed that the class III PI3-kinase Vps34, an important regulator of autophagy, was exclusively up-regulated in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (2.7-fold; p ≤ 10-30). In addition, ectopic expression of ETV6-RUNX1 in cord blood-derived hematopoietic progenitor cells (CB-HPCs) significantly induced expression of Vps34 1.3-fold already 40 hours after transduction (p ≤ 0.05). This suggests that the Vps34-autophagy pathway is activated by ETV6-RUNX1, which may mechanistically explain the leukemogenic and pro-survival properties ascribed to ETV6-RUNX1. In correspondence, Ingenuity Pathway Analysis (IPA) predicted a pro-survival and pro-proliferative phenotype in ETV6-RUNX1 transduced CB-HPCs and highlighted a network of up-regulated transcription factors, including HEY1, EGR1, GATA1 and GATA2 (2 – 25-fold up-regulation; p ≤ 0.05). Luciferase reporter assays revealed that not only the ETV6-RUNX1 fusion protein, but also the ETV6-RUNX1-induced target genes HEY1, EGR1 and GATA1 positively regulate Vps34 promoter activity (5 – 13-fold up-regulation; p ≤ 0.01).Lentiviral knockdown experiments were performed to elucidate the importance of Vps34 expression in ETV6-RUNX1-positive BCP-ALL cells. Knockdown of all Vps34 transcript variants, with two independent constructs, led to complete growth arrest of the ETV6-RUNX1-positive cell lines REH and AT2, while this only led to a decrease in proliferation of the ETV6-RUNX1-negative cell line NALM6. This growth arrest was caused by a significant induction of apoptosis (more than 4-fold 7 days after transduction; p ≤ 0.001) and a significantly reduced percentage of cycling cells (1.3-fold 7 days after transduction; p ≤ 0.05). Analysis of p62 protein expression by western blot and reverse phase protein arrays revealed that the levels of autophagy were significantly higher in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (p ≤ 0.001). In addition, knockdown of ETV6-RUNX1 and Vps34 significantly reduced autophagy, quantified with confocal microscopy, in ETV6-RUNX1-positive cells with 50% and 84%, respectively (p ≤ 0.01). Furthermore, pharmacological inhibition of autophagy with hydroxychloroquine (HCQ) significantly reduced cell viability of BCP-ALL cell lines and primary patient-derived BCP-ALL cells (p ≤ 0.001). Treatment of the ETV6-RUNX1-positive BCP-ALL cell lines REH and AT2 with 20 µg/mL HCQ resulted in a 82% and 95% reduced cell viability, while the viability of ETV6-RUNX1-negative BCP-ALL cell lines and T-ALL cell lines were reduced to a lesser extent (NALM6: 43%; TOM-1: 50%; Loucy: 40%; Jurkat: 0%). Importantly, HCQ selectively sensitized ETV6-RUNX1-positive leukemic cells to L-asparaginase treatment in clinically relevant concentrations. Treatment of primary ETV6-RUNX1-positive patient cells with 10 µg/mL HCQ resulted in a 70% reduction in cell survival during L-asparaginase exposure (p ≤ 0.01). This sensitization was not observed in ETV6-RUNX1-negative BCP-ALL cells. Conclusion: The ETV6-RUNX1 fusion protein activates autophagy via Vps34, which is essential for survival and proliferation of ETV6-RUNX1-positive cells. Inhibition of autophagy in primary ETV6-RUNX1-positive leukemic cells inhibited cell survival and sensitized these cells to L-asparaginase treatment. These results indicate that autophagy inhibition may provide a novel means to sensitize L-asparaginase-resistant ETV6-RUNX1-positive BCP-ALL patients. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Use of native E coli asparaginase in induction leads to high hypersensitivity rates to PEGasparaginase in intensification. Switching to Erwinia asparaginase leads to effective asparaginase activity levels in most patients who experienced an allergy to PEGasparaginase.

Description: Key Points Immune reconstitution after CBT is excellent provided ATG exposure is low or absent. Individualized dosing, or omission of ATG in selected patients, may increase the chance of survival after CBT.

Description: Survival of pediatric acute myeloid leukemia (AML) has improved considerably over the past decades. Since 1985, allogeneic stem cell transplantation (allo-SCT) is widely recommended for patients who have a matched sibling donor. However, it remains controversial whether allo-SCT is superior to chemotherapy for children with newly diagnosed AML. This review summarizes phase 3 clinical trials that compared allo-SCT with chemotherapy (including autologous SCT) in pediatric AML, excluding studies that did not use the intention-to-treat analysis or correct for time-to-transplantation. Although allo-SCT might prevent more relapses than chemotherapy, the number needed for transplantation (with allo-SCT) to prevent one relapse is in the order of 10 patients. Moreover, overall survival is similar with both methods in most recent studies, apparently because of increased salvagability of a relapse when initial therapy concerned chemotherapy only, and because of a higher treatment-related mortality with allo-SCT. Because allo-SCT also gives more severe side effects and results more often in secondary malignancies than chemotherapy, we do not recommend allo-SCT in first remission for pediatric AML in general. Further research should focus on the possibility that subgroups might benefit from allo-SCT, aiming at further improvements in the prognosis of pediatric AML.

Description: Asparaginase is an important component in the treatment of pediatric acute lymphoblastic leukemia (ALL). Erwinia asparaginase is used in patients who develop hypersensitivity to the E. coli derived asparaginase (native asparaginase or PEGasparaginase). Little is known about the pharmacokinetics (PK) of Erwinia asparaginase, especially after intravenous administration. A 100-fold difference in Erwinia asparaginase serum trough levels between patients has been observed during therapeutic drug monitoring (TDM). Currently, in DCOG ALL-protocols dose alterations of asparaginase and erwinase are based on TDM, with the aim to keep the trough asparaginase activity above the 100 IU/L threshold. No formal guidelines for increasing or decreasing the dose are available, and dose-adaptations are based on empirical knowledge. With a PK model, individual dose requirements can be estimated based on their PK parameters like clearance. The final aim is to avoid under-exposure and relapse, or over-exposure which may induce side-effects and results in unnecessary costs. However the upper limit of the therapeutic window that causes toxicity is not known. Aims The aim of this retrospective study was to describe the population PK of Erwinia asparaginase to gather insight in the inter-individual and intra-individual variability, and which co-variates influence exposure. Availability of a population PK model will allow the calculation of a starting dose and subsequent dosing in TDM-setting in the future. Methods A total of 714 evaluable blood samples were collected from 51 children with ALL who received intravenous Erwinia asparaginase as a substitute for E. coli or PEG asparaginase, when treated according to the DCOG ALL-10 or 11 protocol. Patients (32 males and 19 females), had a median age of 6 years (range 1-17), median weight of 25 kg (12-99) and median BSA of 0.92 m2 (0.53-2.22). The median number of samples per patient was 11 (2 - 43). Samples were taken approximately 48 (52.2%) and 72 (36.8%) hours after administration. In addition, samples within 5h (2.4%), between 5-40h (1.3%) and between 80-120h (7.3%) after administration were available. The Erwinia starting dose was 20,000 IU/m2/3x per week, and was given IV over 1 hour, and was later adapted based on TDM to 5,000-50,400 IU/m2 twice (54.2%) or thrice (45.8%) weekly. A population pharmacokinetic model was developed using nonlinear mixed-effects modeling (NONMEM 7.2; pirana 2.7.1; Xpose4). Monte Carlo simulations were performed with different doses and stratification for body weight (n=5000 per dose per weight). Results The concentration versus time profiles were best described with a 2-compartment model with allometric scaling (weight). The parameter estimates were: Cl 0.439 L/h/70kg, V1 (central compartment) 3.22 L/70kg, intercompartimental clearance 0.149 L/h/70kg and V2 (peripheral compartment) 1.14 L/70kg. The interindividual variability of clearance was 32.6%. There was an interoccasion variability of 12.7% based on monthly intervals. Clearance in the first month of treatment was 14% higher compared to the other months (p50kg would have levels 〉100 IU/L after 48h. For patients between 20-50 kg the dose starting dose should be 25,000 IU/m2 in order to achieve levels 〉100 IU/L at 48h in 75% of the patients. In our study, of 36 patients with evaluable starting doses (dosed 20,000 IU/m2 and with an available 48h sample), 38.7% (from a total of 31 patients

Description: BACKGROUND. Allogeneic hematopoietic stem cell transplantation (HSCT) is of benefit in pediatric patients with high-risk acute lymphoblastic leukemia (ALL) in first or further remission. Whether transplantation from unrelated donors could yield similar results to transplantation from HLA identical siblings is still to be assessed within countries running different frontline and relapse protocols. AIM OF THE STUDY. A prospective study was initiated within the International BFM Study Group, in order to assess whether the outcome of HSCT from a 9 or 10 out of 10 HLA allelic matched compatible donor (MD) was inferior to the outcome of HSCT from a matched sibling donor (MSD) in children or young adults with ALL carrying very high risk eligibility criteria for transplantation. Primary endpoint was event-free survival (EFS) and secondary endpoints were non-relapse mortality (NRM) and incidence of acute and chronic graft-versus-host disease (aGVHD, cGVHD). PATIENTS AND METHODS. Between 2007 and 2013, 10 countries (Czech Republic, Denmark, France, Israel, Italy, The Netherlands, Poland, Sweden, Slovakia, Turkey) participated into the ALL SCT I-BFM Study, coordinated by Peters in Vienna. 298 consecutive patients, 18 years old or younger (70% male, median age 9 years), affected with VHR ALL in complete remission (CR), were enrolled in the core arm of the Study (MD vs MSD). Of 298, 107 patients were transplanted from a MSD (50% in CR1, 47% in CR2, 4% 〉CR2) and 191 from a MD (44% in CR1, 48% CR2, 9% 〉CR2), either related (5%) or unrelated (95%), at a median of 189 and 197 days, respectively, after diagnosis/relapse. As per protocol, conditioning regimen consisted of TBI-etoposide, in patients older than 2 years, or busulfan-cyclophosphamide-melphalan, in patients 2 years or younger, and GVHD-prophylaxis consisted of cyclosporine (CSA) only for MSD and CSA/methotrexate/ATG for MD recipients. Median follow-up for alive patients was 3.1 years. RESULTS. Three-year EFS was 67% (SE 5%) for MSD vs 61% (SE 4%) for MD recipients (p-value 0.254), overall survival (OS) 76% (SE 4%) vs 69% (SE 4%) (p-value 0.207), cumulative incidence of relapse (CIR) 24% (SE 4%) vs 22% (SE 3%) (p-value 0.935) and NRM 8% (SE 3%) vs 16% (SE 3%) (p-value 0.094), respectively. There was a trend for a higher risk NRM for MD patients, although no statistical significance was reached (HR 1.94, CI 0.85-4.41; p=0.114), after adjusting for risk profile and donor type. Being transplanted in CR2 was associated with lower EFS and higher NRM (p=0.001). Grade II-IV acute GVHD occurred in 37% and grade III-IV in 16% of MSD vs 42% and 15% of MD recipients; 39% of the evaluable MSD and 19% of the MD recipients experienced chronic GVHD, which was severe in 24% and 10%, respectively. Cumulative incidence of developing chronic GVHD was 39% (SE 5%) and 17% (SE 3%), for patients grafted from MSD and MD, respectively (p=0.001). Being transplanted from a MD, compared with a MSD, was significantly associated with a reduced risk of developing chronic GVHD (HR 0.31, CI 0.18-0.54, p

Description: Key Points miR-139-3p and miR-199a-3p, induced by ICL-induced damage, respectively, cause a loss and gain of hematopoietic progenitors. miR-199a-3p is an onco-microRNA (onco-miR) causing AML in a Cebpa-deficient mouse model. Target genes of miR-199a-3p include PRDX6, RUNX1, and SUZ12.