Structural variants involving MLLT10 fusion are associated with adverse outcomes in pediatric acute myeloid leukemia.

ABSTRACT: MLLT10 gene rearrangements with KMT2A occur in pediatric acute myeloid leukemia (AML) and confer poor prognosis, but the prognostic impact of MLLT10 in partnership with other genes is unknown. We conducted a retrospective study with 2080 children and young adults with AML registered on the Children's Oncology Group AAML0531 (NCT00372593) and AAML1031 trials (NCT01371981). Transcriptome profiling and/or karyotyping were performed to identify leukemia-associated fusions associated with prognosis. Collectively, 127 patients (6.1%) were identified with MLLT10 fusions: 104 (81.9%) with KMT2A::MLLT10, 13 (10.2%) with PICALM::MLLT10, and 10 (7.9%) X::MLLT10: (2 each of DDX3X and TEC), with 6 partners (DDX3Y, CEP164, SCN2B, TREH, NAP1L1, and XPO1) observed in single patients. Patients with MLLT10 (n = 127) demonstrated adverse outcomes, with 5-year event-free survival (EFS) of 18.6% vs 49% in patients without MLLT10 (n = 1953, P < .001), inferior 5-year overall survival (OS) of 38.2% vs 65.7% (P ≤ .001), and a higher relapse risk of 76% vs 38.6% (P < .001). Patients with KMT2A::MLLT10 had an EFS from study entry of 19.5% vs 12.7% (P = .628), and an OS from study entry of 40.4% vs 27.6% (P = .361) in those with other MLLT10 fusion partners. Patients with PICALM::MLLT10 had an EFS of 9.2% vs 20% in other MLLT10- without PICALM (X::MLLT10; P = .788). Patients with PICALM::MLLT10 and X::MLLT10 fusions exhibit a DNA hypermethylation signature resembling NUP98::NSD1 fusions, whereas patients with KMT2A::MLLT10 bear aberrations primarily affecting distal regulatory elements. Regardless of the fusion partner, patients with AML harboring MLLT10 fusions exhibit very high-risk features and should be prioritized for alternative therapeutic interventions.

StarD7 deficiency switches on glycolysis and promotes mitophagy flux in C2C12 myoblasts.

StarD7 is a member of the START protein family required for phosphatidylcholine delivery to the mitochondria, thus key to maintain mitochondrial structure. Its deficiency has been associated with an impairment of cellular processes, such as proliferation and migration, and it has also been reported that it is needed in myogenic differentiation. Here, we show that StarD7 deficiency in C2C12 muscle cells results in the accumulation of abnormal mitochondria, a reduced number of mitochondria per cell area and increased glycolysis. In addition, StarD7-deficient cells undergo an increase in mitochondria-ER contact sites, reduced connexin 43 expression, and disturbances in lipid handling, evidenced by lipid droplet accumulation and decreased levels in phosphatidylserine synthase 1 and 2 expression. Interestingly, StarD7-deficient cells showed alterations in mitophagy markers. We observed accumulation of LC3B-II and BNIP3 proteins in mitochondria-enriched fractions and accumulation of autophagolysosomal and lysosomal vesicles in StarD7-deficient cells. Furthermore, live-cell imaging experiments of StarD7 knockdown cells expressing mitochondria-targeted mKeima indicated an enhanced mitochondria delivery into lysosomes. Importantly, StarD7 reconstitution in StarD7-deficient cells restores LC3B-II expression in mitochondria-enriched fractions at similar levels to those observed in control cells. Collectively, these findings suggest that StarD7-deficient C2C12 myoblasts are associated with altered cristae structure, disturbances in neutral lipid accumulation, glucose metabolism, and increased mitophagy flux. The alterations mentioned above allow for the maintenance of mitochondrial function.

Pathologic, cytogenetic, and molecular features of acute myeloid leukemia with megakaryocytic differentiation: A report from the Children's Oncology Group.

BACKGROUND: Acute myeloid leukemia (AML) with megakaryocytic differentiation (AMkL) is a rare subtype of AML more common in children. Recent literature has identified multiple fusions associated with this type of leukemia. METHODS: Morphology, cytogenetics, and genomic sequencing were assessed in patients from Children's Oncology Group trials AAML0531 and AAML1031 with central-pathology review confirmed non-Down syndrome AMkL. The 5-year event-free survival (EFS), overall survival (OS), and RR were evaluated in these AMkL subcategories. RESULTS: A total of 107 cases of AMkL (5.5%) were included. Distinct fusions were identified in the majority: RBM15::MRTFA (20%), CBFA2T3::GLIS2 (16%), NUP98 (10%), KMT2A (7%), TEC::MLLT10 (2%), MECOM (1%), and FUS::ERG (1%); many of the remaining cases were classified as AMkL with (other) myelodysplasia-related changes (MRC). Very few cases had AML-associated somatic mutations. Cases with CBFA2T3::GLIS2 were enriched in trisomy 3 (p = .015) and the RAM phenotype, with associated high CD56 expression (p < .001). Cases with NUP98 fusions were enriched in trisomy 6 (p < .001), monosomy 13/del(13q) (p < .001), trisomy 21 (p = .026), and/or complex karyotypes (p = .026). While different 5-year EFS and OS were observed in AMkL in each trial, in general, those with CBFA2T3::GLIS2 or KMT2A rearrangements had worse outcomes compared to other AMkL, while those with RBM15::MRTFA or classified as AMkl-MRC fared better. AMkL with NUP98 fusions also had poor outcomes in the AAML1031 trial. CONCLUSION: Given the differences in outcomes, AMkL classification by fusions, cytogenetics, and morphology may be warranted to help in risk stratification and therapeutic options.

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia.

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Malignant Progression of an Ancestral Bone Marrow Clone Harboring a CIC-NUTM2A Fusion in Isolated Myeloid Sarcoma.

Myeloid sarcoma is a rare condition consisting of extramedullary myeloid blasts found in association with acute myeloid leukemia or, in the absence of bone marrow involvement. We identified an infant with isolated myeloid sarcoma whose bone marrow was negative for involvement by flow cytometry. Sequencing revealed the fusion oncogene CIC-NUTM2A and identified the sarcoma to be clonally evolved from the bone marrow, which carried the fusion despite the absence of pathology. Murine modeling confirmed the ability of the fusion to transform hematopoietic cells and identified receptor tyrosine kinase (RTK) signaling activation consistent with disruption of the CIC transcriptional repressor. These findings extend the definition of CIC-rearranged malignancies to include hematologic disease, provide insight into the mechanism of oncogenesis, and demonstrate the importance of molecular analysis and tracking of bone marrow involvement over the course of treatment in myeloid sarcoma, including patients that lack flow cytometric evidence of leukemia at diagnosis. IMPLICATIONS: This study illustrates molecular involvement of phenotypically normal bone marrow in myeloid sarcoma, which has significant implications in clinical care. Further, it extends the definition of CIC-rearrangements to include hematologic malignancies and shows evidence of RTK activation that may be exploited therapeutically in cancer(s) driven by these fusions.

Hypodiploidy has unfavorable impact on survival in pediatric acute myeloid leukemia: an I-BFM Study Group collaboration.

Hypodiploidy, defined as modal numbers (MNs) 45 or lower, has not been independently investigated in pediatric acute myeloid leukemia (AML) but is a well-described high-risk factor in pediatric acute lymphoblastic leukemia. We aimed to characterize and study the prognostic impact of hypodiploidy in pediatric AML. In this retrospective cohort study, we included children below 18 years of age with de novo AML and a hypodiploid karyotype diagnosed from 2000 to 2015 in 14 childhood AML groups from the International Berlin-Frankfurt-Münster (I-BFM) framework. Exclusion criteria comprised constitutional hypodiploidy, monosomy 7, composite karyotype, and t(8;21) with concurring sex chromosome loss. Hypodiploidy occurred in 81 patients (1.3%) with MNs, 45 (n = 66); 44 (n = 10) and 43 (n = 5). The most frequently lost chromosomes were chromosome 9 and sex chromosomes. Five-year event-free survival (EFS) and overall survival (OS) were 34% and 52%, respectively, for the hypodiploid cohort. Children with MN≤44 (n = 15) had inferior EFS (21%) and OS (33%) compared with children with MN = 45 (n = 66; EFS, 37%; OS, 56%). Adjusted hazard ratios (HRs) were 4.9 (P = .001) and 6.1 (P = .003). Monosomal karyotype or monosomy 9 had particular poor OS (43% and 15%, respectively). Allogeneic stem cell transplantation (SCT) in first complete remission (CR1) (n = 18) did not mitigate the unfavorable outcome of hypodiploidy (adjusted HR for OS was 1.5; P = .42). We identified pediatric hypodiploid AML as a rare subgroup with an inferior prognosis even in the patients treated with SCT in CR1.

StarD7 deficiency hinders cell motility through p-ERK1/2/Cx43 reduction.

StarD7 belongs to START protein family involved in lipid traffic, metabolism, and signaling events. Its precursor, StarD7.I which is important for mitochondrial homeostasis, is processed to the StarD7.II isoform that lacks the mitochondrial targeting sequence and is mainly released to the cytosol. StarD7 knockdown interferes with cell migration by an unknown mechanism. Here, we demonstrate that StarD7 silencing decreased connexin 43 (Cx43), integrin ß1, and p-ERK1/2 expression in the non-tumoral migratory HTR-8/SVneo cells. StarD7-deficient cells exhibited Golgi disruption and reduced competence to reorient the microtubule-organizing center. The migratory capacity of StarD7-silenced cells was reestablished when Cx43 level was resettled, while p-ERK1/2 expression remained low. Importantly, ectopic expression of the StarD7.II isoform not only restored cell migration but also ERK1/2, Cx43, and integrin ß1 expression. Thus, StarD7 is implicated in cell migration through an ERK1/2/Cx43 dependent mechanism but independent of the StarD7.I function in the mitochondria.

Ionic complexation improves wound healing in deep second-degree burns and reduces in-vitro ciprofloxacin cytotoxicity in fibroblasts.

The development of new treatments capable of controlling infections and pain related to burns continues to be a challenge. Antimicrobials are necessary tools, but these can be cytotoxic for regenerating cells. In this study, antibiotic-anesthetic (AA) smart systems obtained by ionic complexation of polyelectrolytes with ciprofloxacin and lidocaine were obtained as films and hydrogels. Ionic complexation with sodium alginate and hyaluronate decreased cytotoxicity of ciprofloxacin above 70% in a primary culture of isolated fibroblasts (p < 0.05). In addition, the relative levels of the proteins involved in cell migration, integrin ß1 and p-FAK, increased above 1.5 times (p < 0.05) with no significant differences in cell mobility. Evaluation of the systems in a deep second-degree burn model revealed that reepithelization rate was AA-films = AA-hydrogels > control films > no treated > reference cream (silver sulfadiazine cream). In addition, appendage conservation and complete dermis organization were achieved in AA-films and AA-hydrogels. Encouragingly, both the films and the hydrogels showed a significantly superior performance compared to the reference treatment. This work highlights the great potential of this smart system as an attractive dressing for burns, which surpasses currently available treatments.

Downregulation of krüppel-like factor 6 expression modulates extravillous trophoblast cell behavior by increasing reactive oxygen species.

INTRODUCTION: Placental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated. MATERIALS AND METHODS: KLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated. RESULTS: KLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner. DISCUSSION: Present data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.

Krüppel-like factor 6 participates in extravillous trophoblast cell differentiation and its expression is reduced in abnormally invasive placenta.

Trophoblast cell differentiation is of paramount importance for successful pregnancy. Krüppel-like factor 6 (KLF6), a transcription factor with diverse roles in cell physiology and tumor biology, is required for trophoblast differentiation through the syncytial pathway. Herein, we demonstrate that extravillous trophoblast (EVT) cell migration and mesenchymal phenotype are increased upon KLF6 downregulation or the expression of a deletion mutant lacking its transcriptional regulatory domain (KΔac). Raman spectroscopy revealed molecular modifications compatible with increased differentiation in cells stably expressing the KΔac mutant. Moreover, abnormally invasive placenta showed lower KLF6 immunostaining compared with the normal placenta. Thus, impaired KLF6 expression or function stimulates EVT migration and differentiation in vitro and may contribute to the physiopathology of the abnormally invasive placenta.

Sorafenib in Combination With Standard Chemotherapy for Children With High Allelic Ratio FLT3/ITD+ Acute Myeloid Leukemia: A Report From the Children's Oncology Group Protocol AAML1031.

PURPOSE: High allelic ratio (HAR) FLT3/ITD (AR > 0.4) mutations confer poor prognosis in pediatric acute myeloid leukemia (AML). COG AAML1031 studied the feasibility and efficacy of adding sorafenib, a multikinase tyrosine kinase inhibitor to standard chemotherapy and as single-agent maintenance therapy in this population. MATERIALS AND METHODS: Patients were treated in three cohorts. The initial safety phase defined the maximum tolerated dose of sorafenib starting in induction 2. Cohorts 2 and 3 added sorafenib in induction and as single-agent maintenance. Clinical outcome analysis was limited to n = 72 patients in cohorts 2/3 and compared with n = 76 HAR FLT3/ITD+ AML patients who received identical chemotherapy without sorafenib. Sorafenib pharmacokinetics and plasma inhibitory activity were measured in a subset of patients. RESULTS: The maximum tolerated dose of sorafenib was 200 mg/m2 once daily; dose-limiting toxicities included rash (n = 2; 1 grade 3 and 1 grade 2), grade 2 hand-foot syndrome, and grade 3 fever. Pharmacokinetics/plasma inhibitory activity data demonstrated that measured plasma concentrations were sufficient to inhibit phosphorylated FLT3. Although outcomes were superior with sorafenib in cohorts 2 and 3, patients treated with sorafenib also underwent hematopoietic stem-cell transplant more frequently than the comparator population. Multivariable analysis that accounted for both hematopoietic stem-cell transplant and favorable co-occurring mutations confirmed sorafenib's benefit. Specifically, risk of an event was approximately two-fold higher in HAR FLT3/ITD+ patients who did not receive sorafenib (event-free survival from study entry: hazard ratio [HR] 2.37, 95% CI, 1.45 to 3.88, P < .001, disease-free survival from complete remission: HR 2.28, 95% CI, 1.08 to 4.82, P = .032, relapse risk from complete remission: HR 3.03, 95% CI 1.31 to 7.04, P = .010). CONCLUSION: Sorafenib can be safely added to conventional AML chemotherapy and may improve outcomes in pediatric HAR FLT3/ITD+ AML.

Polygenic Ara-C Response Score Identifies Pediatric Patients With Acute Myeloid Leukemia in Need of Chemotherapy Augmentation.

PURPOSE: To establish a patient-specific polygenic score derived from cytarabine (ara-C) pathway pharmacogenomic evaluation to personalize acute myeloid leukemia (AML) treatment. MATERIALS AND METHODS: Single nucleotide polymorphisms (SNPs) in the ara-C-pathway genes were analyzed with outcome in patients from the multicenter-AML02 trial (N = 166). Multi-SNP predictor modeling was used to develop 10-SNP Ara-C_SNP score (ACS10) using top SNPs predictive of minimal residual disease and event-free survival (EFS) from the AML02-cohort and four SNPs previously associated with ara-C triphosphate levels in the AML97 trial. ACS10 was evaluated for association with outcomes in each clinical trial arms: the standard low-dose ara-C (LDAC, n = 91) and augmented high-dose ara-C (HDAC, n = 75) arms of AML02 and the standard Ara-C, daunorubicin and etoposide (ADE) (n = 465) and the augmented ADE + gemtuzumab ozogamicin (GO; n = 466) arms of AAML0531 trial. RESULTS: In the standard LDAC-arm of AML02 cohort, the low-ACS10 score group (≤ 0) had significantly worse EFS (ACS10 low v high hazard ratio [HR] = 2.81; 95% CI, 1.45 to 5.43; P = .002) and overall survival (OS; HR = 2.98; 95% CI, 1.32 to 6.75; P = .009) compared with the high-ACS10 group (score > 0). These results were validated in the standard-ADE arm of AAML0531, with poor outcome in the low-ASC10 group compared with the high-ACS10 group (EFS: HR = 1.35, 95% CI, 1.04 to 1.75, P = .026; OS: HR = 1.64, 95% CI, 1.2 to 2.22, P = .002). Within the augmented arms (AML02-HDAC and AAML0531-ADE + GO), EFS and OS did not differ between low- and high-ACS10 score groups. In both cohorts, patients with low-ACS10 consistently showed a 10-percentage point improvement in 5-year EFS with augmented therapy (AML02-HDAC or AAML0531-ADE + GO arms) than with standard therapy (AML02-LDAC or AAML0531-ADE arms). CONCLUSION: Patients with low-ACS10 score experienced significantly poor outcome when treated on standard regimen. Augmentation with either high-dose ara-C or GO addition improved outcome in low-ACS10 group. A polygenic ACS10 score can identify patients with unfavorable pharmacogenetic characteristics and offers a potential for an elective augmented therapy option.

Prognostic significance of chromosomal abnormalities at relapse in children with relapsed acute myeloid leukemia: A retrospective cohort study of the Relapsed AML 2001/01 Study.

BACKGROUND: In addition to treatment response, cytogenetic and molecular aberrations are the most important prognostic factors in children with de novo acute myeloid leukemia (AML). However, little is known about cytogenetics at the time of relapse. METHODS: This international study analyzed the prognostic value of cytogenetic profiles and karyotypic changes in pediatric relapsed AML in relation to the probability of event-free (pEFS) and overall survival (pOS). For this purpose, cytogenetic reports from all patients registered on the Relapsed AML 2001/01 Study were reviewed and classified. RESULTS: Cytogenetic information at relapse was available for 403 (71%) of 569 registered patients. Frequently detected aberrations at relapse were t(8;21)(q22;q22) (n = 60) and inv(16)(p13.1q22)/t(16;16)(p13.1;q22) (n = 24), both associated with relatively good outcome (4-year pOS 59% and 71%, respectively). Monosomy 7/7q-, t(9;11)(p22;q23), t(10;11)(p12;q23), and complex karyotypes were associated with poor outcomes (4-year pOS 17%, 19%, 22%, and 22%, respectively). Of 261 (65%) patients for whom cytogenetic data were reliable at both diagnosis and relapse, pEFS was inferior for patients with karyotypic instability (n = 128, 49%), but pOS was similar. Unstable karyotypes with both gain and loss of aberrations were associated with inferior outcome. Early treatment response, time to relapse, and cytogenetic profile at time of relapse were the most important prognostic factors, both outweighing karytoypic instability per se. CONCLUSION: The cytogenetic subgroup at relapse is an independent risk factor for (event-free) survival. Cytogenetic assessment at the time of relapse is of high importance and may contribute to improved risk-adapted treatment for children with relapsed AML.

Cytarabine dose reduction in patients with low-risk acute myeloid leukemia: A report from the Children's Oncology Group.

BACKGROUND: The optimal number of chemotherapy courses for low-risk (LR) pediatric acute myeloid leukemia (AML) is not known. OBJECTIVE: To compare outcomes for four (21.6 g/m2 cytarabine) versus five (45.6 g/m2 cytarabine) chemotherapy courses for LR-AML using data from Children's Oncology Group (COG) AAML0531 and AAML1031. METHODS: We compared relapse risk (RR), disease-free survival (DFS), and overall survival (OS), and the differential impact in LR subgroups for patients receiving four versus five chemotherapy courses. Cox (OS and DFS) and risk (RR) regressions were used to estimate hazard ratios (HR) to compare outcomes. RESULTS: A total of 923 LR-AML patients were included; 21% received five courses. Overall, LR-AML patients who received four courses had higher RR (40.9% vs. 31.4%; HR = 1.40, 95% confidence interval [CI]: 1.06-1.85), and worse DFS (56.0% vs. 67.0%; HR = 1.45, 95% CI: 1.10-1.91). There was a similar decrement in OS though it was not statistically significant (77.0% vs. 83.5%; HR = 1.45, 95% CI: 0.97-2.17). Stratified analyses revealed the detrimental effects of cytarabine dose de-escalation to be most pronounced in the LR-AML subgroup with uninformative cytogenetic/molecular features who were minimal residual disease (MRD) negative after the first induction course (EOI1). The absolute decrease in DFS with four courses for patients with favorable cytogenetic/molecular features and positive MRD was similar to that observed for patients with uninformative cytogenetic/molecular features and negative MRD at EOI1, though not statistically significant. CONCLUSIONS: Our results support de-escalation of cytarabine exposure through the elimination of a fifth chemotherapy course only for LR-AML patients who have both favorable cytogenetic/molecular features and negative MRD after the first induction cycle.

Assessment of Arsenic Trioxide and All-trans Retinoic Acid for the Treatment of Pediatric Acute Promyelocytic Leukemia: A Report From the Children's Oncology Group AAML1331 Trial.

IMPORTANCE: All-trans retinoic acid (ATRA) and arsenic trioxide therapy without the use of maintenance therapy has been found to be beneficial for the treatment of adults with standard-risk acute promyelocytic leukemia (APL). However, it is unclear whether similar regimens are safe and beneficial for the treatment of high-risk APL or pediatric patients with standard-risk APL. OBJECTIVE: To assess whether treatment with an ATRA and arsenic trioxide-based regimen is safe and allows for the elimination or substantial reduction of chemotherapy use among pediatric patients with standard-risk or high-risk APL, respectively. DESIGN, SETTING, AND PARTICIPANTS: The Children's Oncology Group AAML1331 study is a nonrandomized, noninferiority trial that examined survival outcomes among 154 pediatric patients with APL compared with a historical control group of patients with APL from the AAML0631 study. Patients aged 1 to 21 years were enrolled at 85 pediatric oncology centers (members of the Children's Oncology Group) in Australia, Canada, and the US from June 29, 2015, to May 7, 2019, with follow-up until October 31, 2020. All patients had newly diagnosed APL and were stratified into standard-risk APL (white blood cell count <10 000/µL) and high-risk APL (white blood cell count ≥10 000/µL) cohorts. INTERVENTIONS: All patients received ATRA and arsenic trioxide continuously during induction therapy and intermittently during 4 consolidation cycles. Patients with high-risk APL received 4 doses of idarubicin during induction therapy only. The duration of therapy was approximately 9 months, and no maintenance therapy was administered. MAIN OUTCOMES AND MEASURES: Event-free survival (EFS) at 2 years after diagnosis. RESULTS: Among 154 patients (median age, 14.4 years [range, 1.1-21.7 years]; 81 male participants [52.6%]) included in the analysis, 98 patients (63.6%) had standard-risk APL, and 56 patients (36.4%) had high-risk APL. The median follow-up duration was 24.7 months (range, 0-49.5 months) for patients with standard-risk APL and 22.8 months (range, 0-47.7 months) for patients with high-risk APL. Patients with standard-risk APL had a 2-year EFS rate of 98.0% and an overall survival rate of 99.0%; adverse events included 1 early death during induction therapy and 1 relapse. Patients with high-risk APL had a 2-year EFS rate of 96.4% and an overall survival rate of 100%; adverse events included 2 relapses and 0 deaths. These outcomes met predefined noninferiority criteria (noninferiority margin of 10% among those with standard-risk APL and 14.5% among those with high-risk APL). CONCLUSIONS AND RELEVANCE: In this nonrandomized, noninferiority trial, pediatric patients with standard-risk APL who received treatment with a chemotherapy-free ATRA and arsenic trioxide regimen experienced positive outcomes. Patients with high-risk APL also had positive outcomes when treated with a novel ATRA and arsenic trioxide-based regimen that included 4 doses of idarubicin during induction therapy only and no maintenance therapy. The 2-year EFS estimates were noninferior to the historical comparator group, and advantages of the regimen included shorter treatment duration, lower exposure to anthracycline and intrathecal chemotherapy, and fewer days hospitalized. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02339740.

Krüppel-like factor 6 (KLF6) requires its amino terminal domain to promote villous trophoblast cell fusion.

INTRODUCTION: Villous cytotrophoblast (vCTB) cells fuse to generate and maintain the syncytiotrophoblast layer required for placental development and function. Krüppel-like factor 6 (KLF6) is a ubiquitous transcription factor with an N-terminal acidic transactivation domain and a C-terminal zinc finger DNA-binding domain. KLF6 is highly expressed in placenta, and it is required for proper placental development. We have demonstrated that KLF6 is necessary for cell fusion in human primary vCTBs, and in the BeWo cell line. MATERIALS AND METHODS: Full length KLF6 or a mutant lacking its N-terminal domain were expressed in BeWo cells or in primary vCTB cells isolated from human term placentas. Cell fusion, gene and protein expression, and cell proliferation were analyzed. Moreover, Raman spectroscopy and atomic force microscopy (AFM) were used to identify biochemical, topography, and elasticity cellular modifications. RESULTS: The increase in KLF6, but not the expression of its deleted mutant, is sufficient to trigger cell fusion and to raise the expression of ß-hCG, syncytin-1, the chaperone protein 78 regulated by glucose (GRP78), the ATP Binding Cassette Subfamily G Member 2 (ABCG2), and Galectin-1 (Gal-1), all molecules involved in vCTB differentiation. Raman and AFM analysis revealed that KLF6 reduces NADH level and increases cell Young's modulus. KLF6-induced differentiation correlates with p21 upregulation and decreased cell proliferation. Remarkable, p21 silencing reduces cell fusion triggered by KLF6 and the KLF6 mutant impairs syncytialization and decreases syncytin-1 and ß-hCG expression. DISCUSSION: KLF6 induces syncytialization through a mechanism that involves its regulatory transcriptional domain in a p21-dependent manner.

Role of the lipid transport protein StarD7 in mitochondrial dynamics.

Mitochondria are dynamic organelles crucial for cell function and survival implicated in oxidative energy production whose central functions are tightly controlled by lipids. StarD7 is a lipid transport protein involved in the phosphatidylcholine (PC) delivery to mitochondria. Previous studies have shown that StarD7 knockdown induces alterations in mitochondria and endoplasmic reticulum (ER) with a reduction in PC content, however whether StarD7 modulates mitochondrial dynamics remains unexplored. Here, we generated HTR-8/SVneo stable cells expressing the precursor StarD7.I and the mature processed StarD7.II isoforms. We demonstrated that StarD7.I overexpression altered mitochondrial morphology increasing its fragmentation, whereas no changes were observed in StarD7.II-overexpressing cells compared to the control (Ct) stable cells. StarD7.I (D7.I) stable cells were able to transport higher fluorescent PC analog to mitochondria than Ct cells, yield mitochondrial fusions, maintained the membrane potential, and produced lower levels of reactive oxygen species (ROS). Additionally, the expression of Dynamin Related Protein 1 (Drp1) and Mitofusin (Mfn2) proteins were increased, whereas the amount of Mitofusin 1 (Mfn1) decreased. Moreover, transfections with plasmids encoding Drp1-K38A, Drp1-S637D or Drp1-S637A mutants indicated that mitochondrial fragmentation in D7.I cells occurs in a fission-dependent manner via Drp1. In contrast, StarD7 silencing decreased Mfn1 and Mfn2 fusion proteins without modification of Drp1 protein level. These cells increased ROS levels and presented donut-shape mitochondria, indicative of metabolic stress. Altogether our findings provide novel evidence indicating that alterations in StarD7.I expression produce significant changes in mitochondrial morphology and dynamics.

Clinical significance of novel subtypes of acute lymphoblastic leukemia in the context of minimal residual disease-directed therapy.

We evaluate clinical significance of recently identified subtypes of acute lymphoblastic leukemia (ALL) in 598 children treated with minimal residual disease (MRD)-directed therapy. Among the 16 B-ALL and 8 T-ALL subtypes identified by next generation sequencing, ETV6-RUNX1, high-hyperdiploid and DUX4-rearranged B-ALL had the best five-year event-free survival rates (95% to 98.4%); TCF3-PBX1, PAX5alt, T-cell, ETP, iAMP21, and hypodiploid ALL intermediate rates (80.0% to 88.2%); and BCR-ABL1, BCR-ABL1-like and ETV6-RUNX1-like and KMT2A-rearranged ALL the worst rates (64.1% to 76.2%). All but three of the 142 patients with day-8 blood MRD <0.01% remained in remission. Among new subtypes, intensified therapy based on day-15 MRD≥1% improved outcome of DUX4-rearranged, BCR-ABL1-like, and ZNF384-rearranged ALL, and achievement of day-42 MRD<0.01% did not preclude relapse of PAX5alt, MEF2D-rearranged and ETV6-RUNX1-like ALL. Thus, new subtypes including DUX4-rearranged, PAX5alt, BCR-ABL1-like, ETV6-RUNX1-like, MEF2D-rearranged and ZNF384-rearranged ALL have important prognostic and therapeutic implications.