The Effect of Health Insurance on Pediatric Cancer Survival: An Analysis of Children Evaluated for Radiation Therapy in Diverse Multicenter Health Systems.

BACKGROUND: Understanding the role of health insurance in cancer survival in a diverse population of pediatric radiation oncology patients could help to identify patients at risk of adverse outcomes. MATERIALS AND METHODS: Data were collected from cancer patients evaluated for radiation therapy, age < 19, diagnosed from January 1990 to August 2019. Predictors of recurrence-free survival (RFS) and overall survival (OS) were analyzed by univariable and multivariable Cox regression. Variables included health insurance, diagnosis type, sex, race/ethnicity, and socioeconomic status deprivation index. RESULTS: The study included 459 patients with a median diagnosis age of 9 years. Demographic breakdown was 49.5% Hispanic, 27.2% non-Hispanic White, and 20.7% non-Hispanic Black. There were 203 recurrences and 86 deaths observed over a median follow-up of 2.4 years. Five-year RFS was 59.8% (95% CI, 51.6, 67.0) versus 36.5% (95% CI, 26.6, 46.6), and 5-year OS was 87.5% (95% CI, 80.9, 91.9) versus 71.0% (95% CI, 60.3, 79.3) in private pay insurance versus Medicaid/Medicare, respectively. Multivariable showed Medicaid/Medicare patients experienced a 54% higher risk of recurrence (hazard ratio: 1.54, 95% CI, 1.08, 2.20) and 79% higher risk of death (hazard ratio: 1.79, 95% CI, 1.02, 3.14) than privately insured patients. CONCLUSIONS: Significant disadvantages in RFS and OS were identified in radiation oncology patients with Medicaid/Medicare insurance, even after adjusting for clinical and demographic variables.

Late isolated central nervous system relapse in childhood B-cell acute lymphoblastic leukemia treated with intensified systemic therapy and delayed reduced dose cranial radiation: A report from the Children's Oncology Group study AALL02P2.

BACKGROUND: Patients with late, ≥18 months postdiagnosis, isolated central nervous relapse (iCNS-R) of B-acute lymphoblastic leukemia (ALL) have excellent outcomes with chemotherapy plus cranial radiotherapy, with 5-year overall survival (OS) approaching 80% in POG 9412. Subsequent relapse and radiation-related morbidity remain the causes of treatment failure and long-term sequelae. COG AALL02P2 aimed to maintain outcomes in patients with late iCNS-R using intensified chemotherapy and a decrease in cranial irradiation from 1800 to 1200 cGy. PROCEDURES: COG AALL02P2 enrolled 118 eligible patients with B-cell ALL (B-ALL) and late iCNS-R who received intensified systemic therapy, triple intrathecal chemotherapy, and 1200 cGy cranial irradiation delivered at 12 months, with maintenance chemotherapy continuing until 104 weeks postdiagnosis. RESULTS: The 3-year event-free survival (EFS) and OS were 64.3% ± 4.5% and 79.6% ± 3.8%, with 46.1% (18/39) of second relapses including the CNS. Of the 112 patients who completed therapy, 78 received protocol-specified radiation. Study enrollment was closed after interim monitoring analysis showed inferior EFS compared to POG 9412. Patients with initial NCI standard-risk classification fared better than high-risk patients. CONCLUSIONS: COG AALL02P2 showed inferior EFS but similar OS compared to POG 9412. Limitations included a small sample size, more intensive prior therapies, and a significant number of patients (34/118, 29%) who did not receive protocol-directed radiation due to early relapse prior to 1 year or did not otherwise follow the treatment plan. New approaches are needed to improve outcome for these patients and determine the optimal timing and dose of cranial radiation in the treatment of iCNS-R.

A phase I window, dose escalating and safety trial of metformin in combination with induction chemotherapy in relapsed refractory acute lymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEG-asparaginase, and doxorubicin.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains a major cause of death in children. AMP-activated protein kinase (AMPK) affects the unfolded protein response (UPR), leading to increased vulnerability to endoplasmic reticulum (ER) stress in ALL cells. In vitro, metformin causes ALL cell death via AMPK-mediated inhibition of the UPR. It was evaluated whether ER stress could be induced in relapsed ALL through a phase I study investigating the safety and feasibility of metformin in combination with relapse induction chemotherapy. PROCEDURE: Metformin was administered twice daily for 28 days in addition to vincristine, dexamethasone, PEG-asparaginase and doxorubicin (VXLD). Dose escalation of metformin was evaluated using a 3+3 design. Pharmacokinetics (PK), pharmacodynamic (PD) evaluation of the AMPK and ER stress/UPR pathways, and treatment response were assessed. RESULTS: Fourteen patients were enrolled; all were evaluable for toxicity. The recommended phase 2 dose (RP2D) was Dose level 2, 1,000 mg/m2 /day. A single dose-limiting toxicity (DLT), hypoglycemia with acidosis, was observed at the RP2D and two DLTs, diarrhea and acidosis, were observed at Dose Level 3. Nine patients were evaluable for response as defined by the protocol, receiving at least 85% of planned metformin doses. Five complete remissions, one partial response, and one stable disease were observed. PD evaluation showed induction of ER stress, activation of AMPK, and inhibition of the UPR. CONCLUSIONS: The VXLD with metformin was tolerable with a RP2D for metformin of 1,000 mg/m2 /day and yielded responses in a heavily pretreated population. ER stress was induced and toxicities attributable to metformin occurred in all dose levels.

Isolated late testicular relapse of B-cell acute lymphoblastic leukemia treated with intensive systemic chemotherapy and response-based testicular radiation: A Children's Oncology Group study.

BACKGROUND: The incidence of isolated testicular relapse (ITR) of acute lymphoblastic leukemia (ALL) has decreased with contemporary treatment strategies, but outcomes are suboptimal with a 58% 5-year overall survival (OS). This study aimed to improve outcome in patients with ITR of B-cell ALL (B-ALL) occurring after 18 months of first clinical remission using intensive systemic chemotherapy and to decrease long-term sequelae by limiting use of testicular radiation. PROCEDURE: Forty patients in first ITR of B-ALL were enrolled. Induction (dexamethasone, vincristine, daunorubicin, and intrathecal triple therapy) was preceded by one dose of high-dose methotrexate (MTX, 5 g/m2 ). Following induction, 25 of 26 patients who had persistent testicular enlargement underwent testicular biopsy. Eleven had biopsy-proven disease and received bilateral testicular radiation (24 Gy), whereas twenty-nine did not. RESULTS: Overall 5-year event-free survival (EFS)/OS was 65.0 ± 8.8%/73.1 ± 8.3%, with 5-year EFS 62.1 ± 11.0% vs. 72.7 ± 14.4% for patients who did not receive radiation therapy (XRT) (n = 29) compared with those who did (n = 11), respectively (P = 0.64). There were six second bone marrow relapses and six second ITRs. The proportion of second relapses was similar in the patients that received testicular radiation and those who did not. However, the 5-year OS was similar for patients who did not receive XRT (72.6 ± 10.2%) compared with those who did (72.7 ± 14.4%) (P = 0.85). CONCLUSIONS: A 5-year OS rate of 73.1 ± 8.3% was obtained in children with first ITR of B-ALL occurring after 18 months of CR1 (length of first clinical remission) using intensive chemotherapy and limiting testicular radiation.

Adenoid cystic carcinoma of the lacrimal gland in a 14-year-old male.

Adenoid cystic carcinoma (ACC) of the lacrimal gland is an aggressive, malignant epithelial neoplasm. This tumor is rarely seen in adults and even less commonly seen in children and adolescents; thus, there have been no large studies to date describing the optimal treatment of this malignancy in the pediatric population. Here, we report a case of lacrimal gland ACC in a 14-year-old male treated with neoadjuvant intra-arterial chemotherapy followed by globe-sparing tumor resection and chemoradiation. At 2-year follow-up, he remains disease free without evidence of tumor recurrence.

AMPK Associates with Chromatin and Phosphorylates the TAF-1 Subunit of the Transcription Initiation Complex to Regulate Histone Gene Expression in ALL Cells.

The survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL) remain poor. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK). To identify the target genes directly regulated by AMPKα2, we performed genome-wide RNA-seq and ChIP-seq in CCRF-CEM (T-ALL) cells expressing HA-AMPKα2 (CN2) under normal and energy/metabolic stress conditions. CN2 cells show significantly altered AMPKα2 genomic binding and transcriptomic profile under metabolic stress conditions, including reduced histone gene expression. Proteomic analysis and in vitro kinase assays identified the TATA-Box-Binding Protein-Associated Factor 1 (TAF1) as a novel AMPKα2 substrate that downregulates histone gene transcription in response to energy/metabolic stress. Knockdown and knockout studies demonstrated that both AMPKα2 and TAF1 are required for histone gene expression. Mechanistically, upon activation, AMPKα2 phosphorylates TAF1 at Ser-1353 which impairs TAF1 interaction with RNA polymerase II (Pol II), leading to a compromised state of p-AMPKα2/p-TAF1/Pol II chromatin association and suppression of transcription. This mechanism was also observed in primary ALL cells and in vivo in NSG mice. Consequently, we uncovered a non-canonical function of AMPK that phosphorylates TAF1, both members of a putative chromatin-associated transcription complex that regulate histone gene expression, among others, in response to energy/metabolic stress. IMPLICATIONS: Fully delineating the protein interactome by which AMPK regulates adaptive survival responses to energy/metabolic stress, either via epigenetic gene regulation or other mechanisms, will allow the rational development of strategies to overcome de novo or acquired resistance in ALL and other cancers.

Effects of Cancer Treatment on the Education and Cognition of South Florida Pediatric Cancer Survivors.

Purpose As pediatric cancer survival rates have exponentially increased in the past decade, with the vast majority surviving five years or more, the long-term impacts of treatment on the quality of survivorship must be explored. This study examines the effects of pediatric cancer treatment regimens on education outcomes among a demographically diverse regional population. The primary objective is to identify potential factors that may impact the educational and cognitive quality of life in this population.  Methods Four hundred sixty-eight pediatric oncology patients diagnosed at age <20 between January 1990 - August 2019 and treated for cancer with radiation therapy at a large public or a multi-center private hospital in South Florida were identified. A novel survey available in English and Spanish was electronically distributed at least three times to each patient from August 2020 - July 2021 via email, phone call, and text message. Variables relating to demographics, treatment, cognitive impairment, and school re-entry were collected through the survey and electronic medical record review. Descriptive statistical analysis was performed.  Results Of the patients, 10.5% responded to the survey (26 male, 21 female, two unidentified sex). The mean age was 8.9 years old (range 0-20) at diagnosis, 24.0 years old (range 8-39) at the time of survey completion, and 55.1% self-identified as Hispanic. Nearly one-quarter of respondents (22.4%) were unable to correctly identify the treatment modalities they received; Hispanic self-identifying patients were 1.75 times more likely than non-Hispanic patients to incorrectly report the treatment modalities received. One-quarter (26.5%) of respondents reported long-term cognitive deficits post-treatment, of which, over three-quarters (76.9%) identified as Hispanic.  Conclusion This study illuminates patients' perspectives on their long-term cognitive impacts after pediatric cancer treatment. Given the diverse study population, ethnic disparities in post-treatment survivorship were explored. A substantial subset of Hispanic participants was unable to correctly identify their treatment regimen, and a disproportionately large group of Hispanic patients experienced cognitive long-term cognitive deficits, suggesting that ethnic disparities play a critical role in post-treatment survivorship. Further research on prioritizing educational intervention during and after treatment is essential to improving both the quality and equity of survivorship among pediatric oncology patients.

Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG).

BACKGROUND: Sickle-cell anaemia is associated with substantial morbidity from acute complications and organ dysfunction beginning in the first year of life. Hydroxycarbamide substantially reduces episodes of pain and acute chest syndrome, admissions to hospital, and transfusions in adults with sickle-cell anaemia. We assessed the effect of hydroxycarbamide therapy on organ dysfunction and clinical complications, and examined laboratory findings and toxic effects. METHODS: This randomised trial was undertaken in 13 centres in the USA between October, 2003, and September, 2009. Eligible participants had haemoglobin SS (HbSS) or haemoglobin Sß(0)thalassaemia, were aged 9-18 months at randomisation, and were not selected for clinical severity. Participants received liquid hydroxycarbamide, 20 mg/kg per day, or placebo for 2 years. Randomisation assignments were generated by the medical coordinating centre by a pre-decided schedule. Identical appearing and tasting formulations were used for hydroxycarbamide and placebo. Patients, caregivers, and coordinating centre staff were masked to treatment allocation. Primary study endpoints were splenic function (qualitative uptake on (99)Tc spleen scan) and renal function (glomerular filtration rate by (99m)Tc-DTPA clearance). Additional assessments included blood counts, fetal haemoglobin concentration, chemistry profiles, spleen function biomarkers, urine osmolality, neurodevelopment, transcranial Doppler ultrasonography, growth, and mutagenicity. Study visits occurred every 2-4 weeks. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00006400. FINDINGS: 96 patients received hydroxycarbamide and 97 placebo, of whom 83 patients in the hydroxycarbamide group and 84 in the placebo group completed the study. Significant differences were not seen between groups for the primary endpoints (19 of 70 patients with decreased spleen function at exit in the hydroxycarbamide group vs 28 of 74 patients in the placebo group, p=0·21; and a difference in the mean increase in DTPA glomerular filtration rate in the hydroxycarbamide group versus the placebo group of 2 mL/min per 1·73 m(2), p=0·84). Hydroxycarbamide significantly decreased pain (177 events in 62 patients vs 375 events in 75 patients in the placebo group, p=0·002) and dactylitis (24 events in 14 patients vs 123 events in 42 patients in the placebo group, p<0·0001), with some evidence for decreased acute chest syndrome, hospitalisation rates, and transfusion. Hydroxyurea increased haemoglobin and fetal haemoglobin, and decreased white blood-cell count. Toxicity was limited to mild-to-moderate neutropenia. INTERPRETATION: On the basis of the safety and efficacy data from this trial, hydroxycarbamide can now be considered for all very young children with sickle-cell anaemia. FUNDING: The US National Heart, Lung, and Blood Institute; and the National Institute of Child Health and Human Development.

Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury.

Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% +/- 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% +/- 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% +/- 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% +/- 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

Protein Kinase D-Dependent Downregulation of Immediate Early Genes through Class IIA Histone Deacetylases in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and adolescents, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. To identify genome-wide metabolic-stress regulated genes, we used RNA-sequencing in ALL cells treated with AICAR, an AMPK activator. RNA-sequencing identified the immediate early genes (IEGs) as a subset of genes downregulated by AICAR. We show that AICAR-induced IEGs downregulation was blocked by an adenosine uptake inhibitor indicating AICAR was responsible for IEGs reprogramming. Using pharmacologic and genetic models we established this mechanism was AMPK-independent. Further investigations using kinase assays, PKD/PKC inhibitors and rescue experiments, demonstrated that AICAR directly inhibited PKD kinase activity and identified PKD as responsible for IEGs downregulation. Mechanistically, PKD inhibition suppressed phosphorylation and nuclear export of class IIa HDACs, which lowered histone H3 acetylation and decreased NFκB(p65) recruitment to IEGs promoters. Finally, PKD inhibition induced apoptosis via DUSP1/DUSP6 downregulation eliciting a DNA damage response. More importantly, ALL patient cells exhibited the same PKD-HDACs-IEGs-mediated mechanism. As proof of principle of the therapeutic potential of targeting PKD, we established the in vivo relevance of our findings using an NSG ALL mouse model. In conclusion, we identified a previously unreported PKD-dependent survival mechanism in response to AICAR-induced cellular stress in ALL through regulation of DUSPs and IEGs' expression. IMPLICATIONS: PKD mediates early transcriptional responses in ALL cells as an adaptive survival mechanism to overcome cellular stress.

The pediatric hydroxyurea phase III clinical trial (BABY HUG): challenges of study design.

Evidence of the laboratory benefits of hydroxyurea and its clinical efficacy in reducing acute vaso-occlusive events in adults and children with sickle cell anemia has accumulated for more than 15 years. A definitive clinical trial showing that hydroxyurea can also prevent organ damage might support widespread use of the drug at an early age. BABY HUG is a randomized, double-blind placebo-controlled trial to test whether treating young children ages 9-17 months at entry with a liquid preparation of hydroxyurea (20 mg/kg/day for 2 years) can decrease organ damage in the kidneys and spleen by at least 50%. Creation of BABY HUG entailed unique challenges and opportunities. Although protection of brain function might be considered a more compelling endpoint, preservation of spleen and renal function has clinical relevance, and significant treatment effects might be discernable within the mandated sample size of 200. Concerns about unanticipated severe toxicity and burdensome testing and monitoring requirements were addressed in part by an internal Feasibility and Safety Pilot Study, the successful completion of which was required prior to enrolling a larger number of children on the protocol. Concerns over recruitment of potentially vulnerable subjects were allayed by inclusion of a research subject advocate, or ombudsman. Finally, maintenance of blinding of research personnel was aided by inclusion of an unblinded primary endpoint person, charged with transmitting endpoint data and monitoring blood work locally for toxicity (ClinicalTrials.gov number, NCT00006400).

Acute lymphoblastic leukemia mortality in Hispanic Americans.

Higher incidence and poorer outcomes of acute lymphoblastic leukemia (ALL) in Hispanic Americans have been attributed to high-risk molecular markers associated with Native American (NA) ancestry. However, the diverse Hispanic populations in the United States differ substantially in ancestry. Continental Hispanics have a high proportion of NA ancestry while Caribbean Hispanics have a lower proportion of NA ancestry. Here, we analyzed mortality data of 2428 children and adults with ALL. Mortality rates were age-adjusted and compared by race and ethnicity using negative binomial regression with particular attention to distinct Hispanic populations. While both Continental (mortality rate ratio (MRR) 2.09, 95% CI 1.82-2.39) and Caribbean (MRR 1.27, 95% CI 1.05-1.54) Hispanics had higher mortality rates than other racial and ethnic groups, Continental Hispanics had significantly higher mortality rates than Caribbean Hispanics. This is the first study to demonstrate a clear difference in ALL mortality by Hispanic group on a population basis.

Inhibition of the NEDD8 conjugation pathway induces calcium-dependent compensatory activation of the pro-survival MEK/ERK pathway in acute lymphoblastic leukemia.

De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat (TAK-924, MLN4924), a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C ß2 (PKC-ß2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells.

Effect of a formal oncofertility program on fertility preservation rates-first year experience.

BACKGROUND: A formal fertility preservation program was initiated at our institution in 2016 as part of a multi-disciplinary oncofertility initiative to improve the reproductive needs of oncologic patients. After 1 year of initial experience, we assessed sperm banking rates in men diagnosed with cancer, as well as examined the trends in the use of fertility preservation services. METHODS: We performed a chart review from 2011 to 2017 for men newly diagnosed with cancer, and for all men who underwent fertility preservation during that period of time at our institution. We assessed the rates of sperm banking among patients newly diagnosed with cancer, before and after the implementation of a standardized oncofertility program in 2016. The program includes nursing and physician education regarding indications of fertility preservation. Additionally, we evaluated the overall population undergoing sperm cryopreservation at our institution during the study period. RESULTS: From 2011 to 2016, 30 of 902 oncologic patients underwent sperm banking prior to their treatment (3.3% of total cancer patients). After the program was implemented, 42 of 218 patients underwent fertility preservation between June 2016 and August 2017 (19.3% of total cancer patients). In this group, patients' mean age was 30.14 years old (range, 13-69 years old), with 6 pediatric patients; 36 of the samples (85.7%) were obtained from masturbation. When viable sperm could not be obtained from ejaculation, patients underwent either testicular or epididymal sperm extraction (6 cases). Overall, 98 men used the formal fertility preservation service. Of these, 42 were cancer patients and 56 were non-cancer patients. Of the non-cancer patients, 17 banked sperm after varicocelectomy, 6 prior to vasectomy and 6 because of hypogonadism. CONCLUSIONS: Rate of sperm banking increased nearly six-fold after institution of a formal fertility preservation program, indicating the clinical need for such a program at academic institutions. Oncofertility is a relevant part of the care for oncologic patients, and should be considered as part of counseling before cancer treatment.

Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells. RESULTS: We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells. CONCLUSION: AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.

Molecular basis for decreased folylpoly-gamma-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line.

A CCRF-CEM mutant, CEM-p, has been shown to exhibit resistance to methotrexate due to decreased methotrexate polyglutamate accumulation. To ascertain the mechanism(s) responsible for this phenotype, we analyzed FPGS and SLC19A1 mRNA expression, examined FPGS promoter activity, and determined nucleotide sequence of the FPGS promoter and full length cDNA from CCRF-CEM and CEM-p cells. We identified in FPGS from CEM-p cells three amino acid substitutions that altered the ATP binding P-loop, glutamate/folate binding, and a conserved domain located at the carboxyl-terminal. Our data demonstrated for the first time the importance of the highly conserved domain (VTGSLHLVGGV) located at the carboxyl-terminal for FPGS activity.

Analysis of folylpoly-gamma-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells.

BACKGROUND: Expression of folylpoly-gamma-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.

The NEDD8-activating enzyme inhibitor pevonedistat activates the eIF2α and mTOR pathways inducing UPR-mediated cell death in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for adults remain dismal. Further, effective treatment strategies for relapsed/refractory ALL remain elusive. We previously uncovered that ALL cells are prone to apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms. We investigated the antineoplastic activity of pevonedistat®, a novel NEDD8-activating enzyme inhibitor that targets E3 cullin-RING ligases (CRLs) dependent proteasomal protein degradation, in ALL. Herein, we report that pevonedistat induces apoptosis in ALL cells by dysregulating the translational machinery leading to induction of proteotoxic/ER stress and UPR-mediated cell death. Mechanistically, pevonedistat led to P-eIF2a dephosphorylation causing atypical proteotoxic/ER stress from failure to halt protein translation via the UPR and upregulation of mTOR/p70S6K. Additional studies revealed that pevonedistat re-balanced the homeostasis of pro- and anti-apoptotic proteins to favor cell death through altered expression and/or activity of Mcl-1, NOXA, and BIM, suggesting that pevonedistat has a "priming" effect on ALL by altering the apoptotic threshold through modulation of Mcl-1 activity. Further, we demonstrated that pevonedistat synergizes with selected anti-leukemic agents in vitro, and prolongs survival of NSG mice engrafted with ALL cells, lending support for the use of pevonedistat as part of a multi-agent approach.

Primary Mediastinal Large B-Cell Lymphoma With Translocations Involving BCL6 and MYC (Double-Hit Lymphoma).

OBJECTIVES: Primary mediastinal large B-cell lymphomas (PMLBCLs) are aggressive lymphomas with characteristic clinical, morphologic, and immunophenotypic features. "Double-hit" (DH) lymphomas are B-cell neoplasms characterized by a translocation involving MYC and either BCL2 or BCL6 In the indexed literature, there are no reported cases of PMLBCL associated with DH or triple-hit events. METHODS: Herein, we present a case of a 15-year-old girl with PMLBCL who had typical clinical, morphologic, and immunophenotypic features. RESULTS: Fluorescent in situ hybridization studies showed rearrangements involving MYC and BCL6 We also excluded the possibility of a reciprocal t(3;8) (3q27;8q24) BCL6/MYC translocation. CONCLUSIONS: This case expands the current spectrum of lymphomas subtypes in which DH can be found and supports the rationale for cytogenetic testing for DH abnormalities in all cases of aggressive large B-cell lymphomas regardless of subtype.

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress.

BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for ∼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.