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Supplementary Table 1. Antibodies used in the study Figure S1. AICAR-induced downregulation of IEGs mRNA expression in ALL cells. Figure S2. The mechanism of AICAR-induced IEGs downregulation is AMPK-independent. Figure S3. The PKC inhibitor GF109203X suppresses IEGs expression in ALL cells. Figure S4. PKD inhibition downregulates IEGs transcription in ALL cells. Figure S5. The PKD inhibitor CRT synergizes with regorafenib and dexamethasone in ALL cells.

PDF file - 1.3MB, Dose-response of 2-DG and AIX in CCRF-CEM

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.

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.

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PDF file - 707K, Salubrinal does not protect ALL from apoptosis induced by the inhibitors of N-linked glycosylation

PDF file - 240K, Effects of D-mannose on ALL cytotoxicity induced by equimolar concentrations of 2-DG and 2-FDG

PDF file - 415K, Inhibition of mTOR with CCI-779 does not protect ALL cells from 2-DG-cytotoxicity

PDF file - 385K, The AMP analogue AICAr does not significantly affect 2-DG-induced cytotoxicity in ALL cell lines

Background: Panobinostat demonstrates activity against pediatric cancers in vitro. A phase I trial in children with refractory hematologic malignancies was conducted. Study design: The trial evalua...

Background: Patients with late, occurring ≥18 months post-diagnosis, isolated central nervous relapse (iCNS-R) of B-acute lymphoblastic leukemia (ALL) have excellent outcomes with chemotherapy plus cranial radiotherapy, with 5-yr 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-ALL and early iCNS-R who received intensified systemic therapy, triple intrathecal chemotherapy and 1200 cGy cranial irradiation delivered at 12 months, with maintenance chemotherapy continuing until104 weeks post-diagnosis. Results: The 3-yr event-free and overall survival (EFS) and OS were 64.3±4.5% and 79.6±3.8%, with 46.1% (18/39) of 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.

FPBCC was formed in 2018 by five pediatric transplant programs in Florida. One of the key objectives of the consortium is to provide outcome analyses by combining HCT data from all the participating centers in order to identify areas for improvement. In this first FPBCC landscape report we describe the patient and transplant characteristics of pediatric patients undergoing first allo and auto HCT between 2014 and 2016 in Florida. The source of data was eDBtC of the CIBMTR. Over the span of 3 years, a total of 230 pediatric patients underwent allo-HCT and 104 underwent auto-HCT at the participating centers. The most significant predictor of survival in allo-HCT recipients with malignant disorders was the degree of HLA- match, while in the recipients of allo-HCT with non-malignant disorders the predictors of survival included age, donor relationship and degree of HLA match. Our analyses identified the need to improve reporting of primary cause of death and improve on donor selection process given that the degree of HLA match remains the most important predictor of survival. This first FPBCC-wide review describes the trends in pediatric HCT activity between 2014 and 2016 among the participating centers in Florida and confirms feasibility of using eDBtC data platform and collaborative approach in order to identify areas for improvement in outcomes.

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.

Survival rates for relapsed/refractory acute lymphoblastic leukemia (ALL), the most common cancer in children and adolescents, remain dismal. We and others have reported that ALL cells are vulnerable to conditions inducing energy/ER-stress mediated by AMP-activated protein kinase (AMPK) activation. AMPK has been reported to interact with chromatin-associated proteins (e.g., histone H2B) in MEF cells to epigenetically regulate gene expression in response to environmental/cellular stress (Bungard et al. Science, 2010; 329:1201). To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we first constructed Bp-ALL (NALM6, REH) and T-ALL (CCRF-CEM, KE-37) stable cell lines expressing HA-AMPKα1 or HA-AMPKα2. Next, using HA and RNA pol II antibodies, we performed ChIP-seq assays in CCRF-CEM/HA-AMPKα2 (CN2) grown in glucose-free RPMI for 24 h. ChIP-seq differentially identified 171 candidate genes in CN2 treated with no-glucose vs. 431 genes in untreated controls. Data analysis using the Encode and ChEA database identified the TATA-Box Binding Protein Associated Factor, CCAAT Enhancer Binding Protein Delta (CEBPD), the negative elongation factor complex member E (NELFE), and the Promyelocytic leukemia protein (PML) among highly ranked transcription factors (TFs) may associated with AMPKα2 on chromatin. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin gene loci regulated in response to energy/metabolic stress, we performed RNA-seq assays in CN2 cells treated with or without glucose deprivation for 24 h. RNA-seq data analysis indicated that of the 3497 genes altered by AMPK activation, two thirds were downregulated whereas the remaining were upregulated. Kyoto Encyclopedia of Genes and Genomes gene set and BioPlanet 2019 gene set analysis identified metabolic pathways, DNA replication/metabolism, and cell cycle as the main biological processes altered in CN2 cells in response to metabolic stress. Among downregulated genes in response to metabolic stress, we uncovered a cluster of histone genes. To confirm and validate our data, we used RT-qPCR and ChIP-qPCR assays on selected histone gene candidates (H1-2/ HIST1H1C, H1-3/HIST1H1D, H4C4/HIST1H4D) which exhibited both decreased recruitment of HA-AMPKα2 to chromatin and mRNA downregulation in response to metabolic stress. Further ChIP-qPCR assays using an AMPKα2 antibody confirmed these data in KASUMI-2 cells (Bp-ALL). Similar data were also observed in CN2 cells treated with AICAR, another AMPK activator. Additional experiments were conducted using the allosteric AMPK activators compound 991 and PF-06409577. Using Co-IP experiments, we uncovered that AMPKα2 interacted with putative members of an AMPK/chromatin-associated transcription factor complex which included the TATA-Box Binding Protein Associated Factor (TAF), integrator (INT), and RNA polymerase II. To investigate the role of AMPK kinase activity on AMPKα2-associated chromatin regulated gene targets, we determined the effect of genetic constructs encoding a constitutively active (CA) form of AMPKα2 on histone gene mRNA expression in CCRF-CEM and MEF AMPKα1/AMPKα2 double knockout (DKO) cells, and found that in both cell types the expression of the full-length CA-AMPKα2 (T172D) lead to decreased mRNA expression of the histone genes examined, suggesting AMPK kinase activity is required to regulate histone genes in response to energy/metabolic stress. In conclusion, our data show that in response to metabolic stress, AMPK binds directly to a multi-protein complex on chromatin to reprogram gene expression to promote cellular adaptation/survival in ALL. Further elucidation of AMPK's interactions with members of the putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions and combination strategies to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies. Disclosures No relevant conflicts of interest to declare.

Unregulated growth and replication as well as an abnormal microenvironment, leads to elevated levels of stress which is a common trait of cancer. By inducing both energy and endoplasmic reticulum (ER) stress, 2-Deoxy-glucose (2-DG) is particularly well-suited to take advantage of the therapeutic window that heightened stress in tumors provides. Under hypoxia, blocking glycolysis with 2-DG leads to significant lowering of ATP resulting in energy stress and cell death in numerous carcinoma cell types. In contrast, under normoxia, 2-DG at a low-concentration is not toxic in most carcinomas tested, but induces growth inhibition, which is primarily due to ER stress. Here we find a synergistic toxic effect in several tumor cell lines in vitro combining 2-DG with fenofibrate (FF), a drug that has been safely used for over 40 years to lower cholesterol in patients. This combination induces much greater energy stress than either agent alone, as measured by ATP reduction, increased p-AMPK and downregulation of mtor. Inhibition of mtor results in blockage of GRP78 a critical component of the unfolded protein response which we speculate leads to greater ER stress as observed by increased p-eif2 alpha. Moreover, to avoid an insulin response and adsorption by the liver, 2-DG is delivered by slow-release pump yielding significant anti-tumor control when combined with FF. Our results provide promise for developing this combination clinically and others that combine 2-DG with agents that act synergistically to selectively increase energy and ER stress to a level that is toxic to numerous tumor cell types., NA

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.

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.

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 β2 (PKC-β2) 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.

Background Graft vs Host Disease (GVHD) is a common but revered complication after allogenic hematopoietic stem cell transplants (HSCT). Corticosteroids are the first-line therapy for GVHD, yet a consensus is lacking for second-line therapy in patients who experience worsening of symptoms when doses are reduced after initial response (steroid dependent), or who progress despite optimum steroid therapy (steroid refractory) GVHD. Bortezomib is a first-generation reversible proteasome inhibitor that inhibits T cells and prevents activation of dendritic cells that mediate antigen presentation and cytokine transcription. Though promising in adult clinical trials, there is a paucity of data on children less than 18 years for the prevention and treatment of GVHD. We hereby report on a single institutional case-series of bortezomib in pediatric HSCT treatment of GVHD. Results Our first case, a 7 year old male with history of acute myeloid leukemia, developed stage 1 acute gastrointestinal GVHD 23 days post-HSCT, and stage 4 skin GVHD beginning 39 days post-HSCT. His skin GVHD failed multiple attempts to wean off steroids, hence the addition of bortezomib at 364 days post-HSCT, receiving weekly doses for 30 weeks ranging from 0.5 mg/m2 to 1.0 mg/m2. This allowed us to wean him off prednisone and discontinue all immune suppression without additional flares. Our second case, a 4 year old male, presented with acute stage 4 skin and stage 2 liver GVHD 108 days and 341 post-HSCT, respectively. Bortezomib was started for the skin GVHD after unable to wean him off steroids. He received a total of 15 doses administered weekly and ranging from 0.4 to 0.87 mg/m2. While on bortezomib his skin GVHD resolved allowing discontinuation of steroids and other immune-suppressive agents. He later developed liver GVHD, which was not responsive to bortezomib. Our third case, a 1 year old female, presented with steroid-dependent acute skin GVHD 177 days post-HSCT. She responded remarkably to steroids but experienced multiple flares of skin GVHD any time the steroid dose was reduced below 1 mg/kg/day. After multiple attempts employing different combinations of immunosuppressive regimens, bortezomib was started. She showed initial response to bortezomib at doses ranging from 0.1 mg to 1.3/m2 mg but was unable to be completely weaned off steroids. Our fourth case, a 19 year old male with lung GVHD who was on long-term steroids, received bortezomib after failing steroid dose reduction when combined with sirolimus and extra-corporeal photopheresis. He received weekly doses of bortezomib for 12 weeks, ranging from 0.2 mg to 1.3/m2 mg, that permitted a successful wean off steroids. However, he later died from pulmonary GVHD. Conclusion We hereby report, based on these cases that bortezomib is a safe option for adjuvant GVHD therapy in children after HSCT. It resulted in successful discontinuation of steroids in two of the three patients with skin GVHD. Well-designed studies of GVHD management with bortezomib in pediatrics are necessary to elucidate this initial finding. We plan to explore this further in a multi-center trial. OffLabel Disclosure: Bortezomib is off label - FDA indications are for treatment of multiple melanoma and second line mantel cell lymphoma

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. We previously uncovered that ALL are vulnerable to metabolic/energy stress and ER-stress via AMP-activated protein kinase (AMPK) activation. In order to identify genome wide metabolic stress and AMPK transcriptionally regulated genes, we used RNA-Seq and compared mRNA profiles in ALL cells treated with the adenosine analog AICAR, an activator of AMPK. RNA-Seq data indicated that high dose AICAR (15 mM/45 min) induced a robust downregulation of a cluster of genes known as the immediate early genes (IEGs), which are critical for cell survival, proliferation and adaptation. AICAR-induced downregulation on IEG expression was dose- and time-dependent, and observed in other cell types (HEK293T, Hela, MEF), indicating this mechanism is conserved in mammalian cells. Using MEF AMPKα2 and AMPKα1/α2 knockout cell lines, we found that these alterations were AMPK-independent. Characterization of AICAR’s mechanism of action identified the protein kinase D1 (PKD1) as responsible for these effects. PKD1 is a Ser/Thr protein kinase involved in many cellular processes important for cancer development and progression, including proliferation, survival, apoptosis, invasion, cell adhesion and angiogenesis. We uncovered that high dose AICAR significantly inhibited PKD1 activation (Ser-910) at the plasma membrane which prevented its nuclear translocation. When PKD1 activity was pharmacologically inhibited by CRT0066101 or downregulated by shRNA, we observed similar IEGs’ downregulation in ALL cells. Conversely, the effect of AICAR on IEGs’ expression was abrogated by PMA, a direct activator of PKD1. In addition, when PKD1 was overexpressed in HEK293T cells, AICAR-induced IEG’s downregulation was partially restored. Using a kinase assay, we found that AICAR, but not ZMP, directly inhibited PKD1 kinase activity. Further, we determined that AICAR suppressed phosphorylation and nuclear export of PKD1-targeted histone deacetylases HDAC4/5, which led to decreased histone H3 acetylation at the IEGs’ promoter region. Finally, ChIP-qPCR indicated that AICAR-induced PKD1 inhibition prevented NF-κB recruitment to IEGs’ promoters. Inhibition of PKD1 activity led to decreased cell proliferation and promoted apoptosis in ALL cells. To confirm the in vivo relevance of our data, single agent and combination experiments using our NSG ALL mouse model are underway. Taken together, we have identified a novel AMPK-independent mechanism leading to AICAR’s inhibition of PKD1-mediated ALL survival. Consequently, co-targeting PDK1 and other pro-survival stress response pathways in ALL cells offers novel strategies to overcome therapeutic resistance. Citation Format: Anna Shvab, Guangyan Sun, Bin Li, Felipe Beckedorff, Guy J. Leclerc, Ramin Shiekhattar, Julio C. Barredo. AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1289.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for relapsed/refractory ALL in children and adults remain dismal, highlighting the need for novel targeted therapies capable of overcoming resistance in relapsed/refractory disease. We previously uncovered that ALL cells are vulnerable to metabolic/energy stress and endoplasmic reticulum (ER)-stress via AMP-activated protein kinase (AMPK) activation leading to unfolded protein response (UPR)-mediated apoptosis. In order to identify genome-wide metabolic-stress and AMPK-transcriptionally regulated genes in ALL cells undergoing metabolic/energy stress, we used RNA-Seq and compared mRNA transcript profiles in ALL cells treated with acadesine (adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or AICAR), known to activate AMPK. RNA-Seq data indicated that acadesine treatment (15 mM/45 min) induced a robust and rapid alteration in gene expression in ALL cells. The most significant acadesine-induced gene signature represented a cluster of genes known as immediate early genes (IEGs), which are fundamental in critical biological pathways for cell survival/proliferation/adaptation. We interpreted these changes as a compensatory pro-survival mechanism in ALL cells undergoing energy/metabolic stress. Among the acadesine-induced downregulated IEGs, we selected DUSP1, JUNB and NFKBIA for further characterization. Downregulation of these IEGs was confirmed using RT-qPCR. We found that the effect of acadesine-induced downregulation on IEGs expression was dose- and time-dependent, and these effects were observed in other cell types (HeLa, HEK293T, mouse embryonic fibroblasts(MEF)), indicating this mechanism of acadesine-induced downregulation of IEGs expression is conserved in mammalian cells. Interestingly, when we used lower doses of acadesine (the half-maximal inhibition concentration for IEGs (IC50)), the IEGs mRNA levels returned to baseline after 3 hours of exposure, suggesting the effect of acadesine on these IEGs was transient at IC50 dose. Using NALM6 AMPKα1 knockdown and MEF AMPKα1/α2 knockout cell lines, we uncovered that high-dose/short-time exposure to acadesine led to changes in IEGs expression that were independent of AMPK. Consistent with these findings, ALL cells co-treated with acadesine plus adenosine kinase inhibitors (ABT702 or 5-Iodotubercidin), which prevent its conversion to ZMP, exhibited the same gene expression signature. Characterization of acadesine's mechanism of action identified protein kinase D1 (PKD1) as responsible for acadesine-induced downregulation of IEGs. PKD1 is a serine/threonine protein kinase involved in many cellular processes important to cancer development and progression, including proliferation, survival, apoptosis, motility, cell adhesion and angiogenesis. Acadesine induced strong inhibition of PKD1 activity which resulted in PKD1 accumulation in the cytoplasm and prevented its nuclear translocation. When ALL cells were treated with protein kinase D (PKD) inhibitors (CRT0066101, GF109203X), we observed a similar rapid, robust and transient downregulation of IEGs, suggesting acadesine interacts with the PKD1 pathway. Conversely, the effect of acadesine on IEGs expression was abrogated by phorbol 12-myristate 13-acetate (PMA), a direct activator of PKD. Further, we determined that acadesine suppresses PKD1-regulated class II Histone deacetylase (HDAC4/5) phosphorylation and nuclear export, which led to decreased histone H3 acetylation levels at the IEG's promoter region. Finally, ChIP-qPCR experiments uncovered that the acadesine/PKD1 axis regulates the recruitment of nuclear factor-κB (NF-κB) to the promoter region of selected IEGs. Consequently, we have identified a novel, AMPK-independent transcription regulation mechanism of acadesine thorugh PKD1 in ALL cells, and co-targeting PDK1 and other pro-survival stress response pathways in ALL cells vulnerable to energy/metabolic stress offers potential novel strategies to overcome therapeutic resistance. Disclosures No relevant conflicts of interest to declare.

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.

The central nervous system (CNS) has long been recognized as a site, indeed a sanctuary, for leukemic cells. Although few (

Folate cofactors are essential components of one carbon metabolism and are required for the biosynthesis of purines, pyrimidines, serine and methionine. The classical folate antagonist methotrexate (MTX) continues to be a universal component of most ALL treatment regimens. MTX is retained within cells as long-chain polyglutamates (MTX-PGs) after metabolism by the enzyme folylpoly-γ -glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). The FPGS gene is regulated by the transcription factors NFY and Sp1. Using DNaseI assays we identified a hypersensitive site mapping closely upstream of exon 1, suggesting that chromatin remodeling may contribute to FPGS gene regulation. To investigate the role of histone modifications and chromatin remodeling on FPGS expression and uncover interactions between NFY, Sp1 and HDAC1, we performed co-immunoprecipitation and Western blotting. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assays (DAPA) revealed that HDAC1 is recruited by NFY and Sp1 to the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by NFY and Sp1 factors interacting with HDAC1, and leading to chromatin remodeling. We then examined the effect of the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaBu) and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes in NALM6 (Bp-ALL), REH (TEL/AML1+, Bp-ALL), SupB15 (BCR/ABL+, Bp-ALL), and CCRF-CEM (T-ALL) cells using qRT-PCR. In all cell lines examined, treatment with HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas the level of DHFR and TS mRNA expression were decreased. On this basis, we hypothesized that induction of FPGS expression by HDACIs, results in higher accumulation of MTX-PG and enhanced MTX cytotoxicity in ALL cells. Further, the concomitant decrease in the expression of the MTX-PG target enzymes DHFR and TS, would enhance the cytotoxicity of the combination of HDACIs plus MTX in ALL cells. To test this hypothesis, NALM6, REH, and SupB15 cells were treated with MTX (4h) + SAHA (24h), and cell viability assessed. We determined that SAHA increased the intracellular accumulation of long chain MTX-PGs (n ≥3 Glu) in ALL cells, correlating with the upregulation of FPGS expression in SAHA-treated cells. Treatment with MTX + SAHA increased cytotoxicity by ~30% with a calculated combination index of ≤ 0.8 indicating synergy. Analysis of apoptosis using AnnexinV/PI staining revealed a 2 to 3-fold increase in apoptotic cell death in all cell lines treated with this combination. Our data suggest HDACIs enhance MTX cytotoxicity by upregulation of FPGS expression, increased accumulation of MTXPG and downregulation of DHFR and TS. The synergism exhibited by the combination of MTX and SAHA suggests it should be tested in ALL patients, in particular those who exhibit phenotypes with de novo or acquired resistance to MTX.

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). Pediatr Blood Cancer 2010;54:250–255. © 2009 Wiley-Liss, Inc.

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.

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.

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.

Although improvements in the management of sickle cell disease (SCD) have increased patient survival into adulthood, morbidity and mortality from end-organ damage remain major concerns. One of the most serious complications of SCD is renal failure, affecting about 20% of patients. The clinical manifestations of sickle cell nephropathy (SCN) involve changes in glomerular ultrastructure, albuminuria, and a progressive decline in glomerular hemodynamics. The mechanisms or factors that promote SCN are not fully elucidated. In the present study, the role of renal kallikrein as a risk marker for promoting SCN was explored in a cross-sectional study.We measured the urinary excretion rate of active kallikrein in 73 children with sickle cell anemia (hemoglobin SS, SC, or S thalassemia) and in 30 control healthy African American children. The findings demonstrated that a significant difference in the excretion rate of log kallikrein in male versus female patients with SCD, P0.0078 was observed. In children with SCD, cross-sectional analysis revealed a positive and significant correlation between the excretion rate of active kallikrein and log albumin excretion rate (AER), P0.0088. Regression analysis also determined that the excretion rate of active kallikrein negatively correlates with hemoglobin in children with SCD, P0.0096. In addition, an inverse relationship between log AER and hemoglobin was observed in male patients with SCD, P0.0143. In children with SCD, cross-sectional analysis revealed a positive and significant correlation between log AER and age, suggesting age as a risk marker for AER in SCD. In multivariate regression analysis, our findings demonstrate a strong association between log AER and age and log kallikrein in children with SCD. About 20% of the variability in log AER in SCD patients is influenced by age and 6% is influenced by log kallikrein, P0.0001 and P0.02, respectively.These findings provide the first evidence that the excretion rate of active kallikrein is positively and independently correlated with log AER in children with SCD, and suggest that kallikrein could be a marker for progressive nephropathy. Longitudinal studies are essential to address this issue.

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.

Background: Relapsed Acute Lymphoblastic Leukemia (ALL) remains a major cause of cancer-related deaths in children. We identified the AMP activated protein kinase (AMPK) as a potential target for ALL therapy due to its regulatory effects on the unfolded protein response (UPR), leading to increased vulnerability of ALL cells to endoplasmic reticulum (ER) stress inducers. In vitro, metformin leads to ALL cell death via AMPK-mediated inhibition of the UPR. Methods: Metformin was administered twice daily continuously on a 28 day cycle in addition to the Vincristine, Dexamethasone, PEG-Asparaginase and Doxorubicin (VPLD) systemic regimen and CNS-directed therapy in pediatric patients with relapsed/refractory ALL. Metformin doses were increased in a standard 3+3 phase I design with three dose levels evaluated, 666, 1,000 and 1,333 mg/m2/day. Pharmacokinetic (PK) and pharmacodynamic (PD) evaluation of the AMPK and ER stress/UPR pathways were ascertained on days 1 and 7, and treatment response was assessed on day 29. Results: Fourteen patients were enrolled, 11 evaluable. DL3 was the maximum administered dose with 2 related DLT’s of diarrhea and acidosis. A single DLT of hypoglycemia and acidosis during an episode of sepsis was observed in DL2. Infectious SAE’s occurred in 7 patients. Two patients had posterior reversible encephalopathy syndrome; both died of disease progression within 30 days of coming off study. A single patient had stable disease, 2 had a partial response, and 3 achieved a complete response. PK studies demonstrated levels within the therapeutic range for patients with diabetes, and PD evaluation showed induction of ER stress and inhibition of the UPR. Conclusions: This trial has been completed. We found induction of ER stress with inhibition of UPR consistent with that observed in vitro leading to metformin-induced apoptosis. The chemotherapeutic backbone was tolerable and the combination with metformin yielded responses in a heavily pretreated population. Toxicities attributable to metformin occurred in all dose levels, but DLT’s were only observed in dose levels above the standard dosing for diabetes. Clinical trial information: NCT01324180 Citation Format: Matteo Trucco, Julio Barredo, John Goldberg, Gregory Hale, Jonathan Gill, Bhuvana Setty, Tiffany Smith, Jae Lee, Damon Reed. A phase I window, dose escalating and safety trial of Metformin in combination with induction chemotherapy (VPLD)in relapsed/refractory acute lymphoblastic leukemia: NCT01324180 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT145. doi:10.1158/1538-7445.AM2017-CT145

PURPOSE To describe a patient with retinoblastoma and morning glory disk anomaly (MGDA). METHOD Observational case report. RESULTS A 7-month-old girl with bilateral retinoblastoma was found to have concurrent MGDA in the less involved eye. The more severely affected eye was enucleated due to progressive disease not amenable to aggressive systemic and local treatment; histologic analysis was performed. CONCLUSION Prompt ocular examination of a pediatric patient should be performed in the presence of concerning findings such as leukokoria or strabismus. In addition, more than one ocular abnormality may coexist, such as retinoblastoma and MGDA, necessitating complete examination.

Contemporary chemotherapy has significantly improved event-free survival among patients with T cell-lineage acute lymphoblastic leukemia (T-ALL). Unlike B-precursor ALL, most investigators are still using cranial radiation (CRT) and are hesitant to rely solely on intrathecal therapy for T-ALL. In this study we assessed the effects of CRT upon event-free survival and central nervous system (CNS) relapses in a cohort of children with high risk features of T cell leukemia. In a series of six consecutive studies (1987-1995) patients were non-randomly assigned their CNS prophylaxis per individual protocol. These protocols were based on POG 8704 which relied on rotating drug combinations (cytarabine/cyclophosphamide, teniposide/Ara-C, and vincristine/doxorubicin/6-MP/prednisone) postinduction. Modifications such as high-dose cytarabine, intermediate-dose methotrexate, and the addition of G-CSF, were designed to give higher CNS drug levels (decreasing the need for CRT), to eliminate epidophyllotoxin (decreasing the risk of secondary leukemia), and to reduce therapy-related neutropenia (pilot studies POG 9086, 9295, 9296, 9297, 9398). All patients included in this analysis qualified for POG high risk criteria, WBC >50000/mm3 and/or CNS leukemia. Patients without CNS involvement received 16 doses of age-adjusted triple intra-thecal therapy (TIT = hydrocortisone, MTX, and cytarabine) whereas patients with CNS disease received three more doses of TIT during induction and consolidation. Patients who received CRT were treated with 2400 cGy (POG 8704) or 1800 cGy (POG 9086 and 9295). CNS therapy included CRT in 144 patients while the remaining 78 patients received no radiation by original protocol design. There were 155 males and 57 females with a median age of 8.2 years. The median WBC for the CRT+ and CRT- patients were 186000/mm3 and 200000/mm3, respectively. CNS involvement at diagnosis was seen in 16% of the CRT+ and 23% of the CRT- groups. The complete continuous remission rate (CCR) was not significantly different for the irradiated vs. non-irradiated groups (P = 0.46). The 3-year event-free survival was 65% (s.e. 6%) and 63% (s.e. 4%) for the non-irradiated vs. the radiated group. However, the 3-year CNS relapse rate was significantly higher amongst patients who did not receive CRT; 18% (s.e. 5%) vs. 7% (s.e. 3%) in the irradiated group (P = 0.012). Our analysis in a non-randomized setting, suggests that CRT did not significantly correlate with event-free survival but omitting it had an adverse effect on the CNS involvement at the time of relapse.

Immunophenotype might be an important indicator for multidrug resistance (MDR) profiles in childhood acute lymphoblastic leukemia (ALL). The authors analyzed the messenger RNA (mRNA) levels of MDR1, multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP) by reverse transcriptase-polymerase chain reaction (RT-PCR) in childhood pre-B ALL, T-cell ALL, and acute nonlymphoblastic leukemia (ANLL). Results showed that MRP and LRP, but not MDR1, mRNAs are overexpressed, particularly in children with pre-B ALL compared with T-cell ALL and ANLL tested. In addition, the MRP and LRP mRNA expression levels in initial diagnosis and first relapse samples of one patient with pre-B ALL were similar. Consequently, these preliminary results suggest that the expression of these MDR-related genes in childhood ALL might be regulated differently in a lineage dependent manner.

The effect of down-regulation of folylpoly-γ-glutamate synthetase (FPGS) activity on intracellular reduced folate accumulation and cellular proliferation was examined, using an inducible antisense expression system in the human T-lymphoblastic leukemia cell line CCRF-CEM. FPGS catalyzes the addition of γ-glutamyl residues to natural folates and classical antifolates, which results in their enhanced cellular retention and increased cytotoxicity. As such, this enzyme has become a focus as a potential anticancer drug target. However, direct evidence to support this concept has been elusive. Hence, a study was undertaken using an antisense-based expression system to down-regulate FPGS activity. This inducible expression system was used to demonstrate that lower FPGS activity can lead to substantially lower intracellular folate content, which coincides with suppression of thymidylate synthesis and inhibition of cellular proliferation.

Background We present and discuss the successful treatment of pleuropulmonary blastoma metastatic to the brain using a multimodality regimen with surgery, high-dose chemotherapy and radiation therapy. Procedure A 3-year-old boy referred to our institution with bilateral pulmonary cysts was diagnosed with pleuropulmonary blastoma (PPB). Initial treatment included surgery and multiagent chemotherapy with vincristine, dactinomycin, cyclophosphamide, cisplatin, and doxorubicin. One year after the completion of therapy, his PPB recurred as an intracerebral metastasis, and required further treatment with a multimodality salvage regimen. The child was successfully treated with a subtotal surgical resection, followed by high-dose cyclophosphamide, and radiation therapy. He is now disease-free 24 months later. Results Intracerebral metastases of PPB have been a uniformly fatal complication of this tumor. Postsurgical chemotherapy and radiation therapy appear to have contributed to the prolonged survival and potential for cure in our patient. Conclusions The use of this multimodality regimen may be warranted in other patients with recurrent PPB metastatic to the brain. Med. Pediatr. Oncol. 30:63–66, 1998. © 1998 Wiley-Liss, Inc.

Purpose The treatment of infant acute lymphoblastic leukemia (ALL) continues to be a significant challenge for pediatric oncologists due to the high incidence of early relapses. Salvage regimens used to date have met limited success. We describe two cases of relapsed infant ALL who have achieved long-term survival with an intensive antimetabolite-based salvage regimen. Patients and Methods Two consecutive infants with relapsed ALL presented at our institution and were treated with an antimetabolite-based regimen. Both cases exhibited clinical and biological phenotypes previously associated with infantile ALL. Results Both patients have achieved prolonged and sustained remissions 48 and 30 months EFS respectively following therapy with intensive antimetabolite-based salvage regimen. Conclusions An intensive multiagent antimetabolite-based salvage regimen resulted in prolonged EFS in two cases of relapsed infant ALL. Dose intensification was achieved by administering repeated cycles of the same treatment schema using high dose chemotherapy throughout therapy. These infants were spared prophylactic cranial irradiation without a negative impact on outcome. The use of L-asparaginase, timed after high-dose Cytarabine (ARA-C) throughout therapy, might have contributed to their cure. Med. Pediatr. Oncol. 29:256–259, 1997. © 1997 Wiley-Liss, Inc.

Cellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B-lineage ALL (129 children) compared with those with T-lineage ALL (20 children) (p < 0.01) and was characterized by a saturable (Emax) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 microM), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (Emax in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM6 cells, and v) 4-6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC50) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.

OBJECTIVE: To catalog and evaluate patterns of disease in families of children with pleuropulmonary blastoma (PPB). METHODS: Data have been collected since 1988 on 45 children with PPB and their families. All pathologic materials were centrally reviewed. Preliminary molecular genetic analyses were performed when possible. RESULTS: In 12 of 45 patients, an association was found between PPB and other dysplasias, neoplasias, or malignancies in the patients with or in their young relatives. The diseases found to be associated with PPB include other cases of PPB, pulmonary cysts, cystic nephromas, sarcomas, medulloblastomas, thyroid dysplasias and neoplasias, malignant germ cell tumors, Hodgkin disease, leukemia, and Langerhans cell histiocytosis. Abnormalities of the p53 tumor suppressor gene, Wilms tumor suppressor gene (WT1), and the putative second genetic locus for Wilms tumor (WT2) were not found in preliminary investigations. CONCLUSIONS: The occurrence of PPB appears to herald a constitutional and heritable predisposition to dysplastic or neoplastic disease in approximately 25% of cases. All patients with PPB and their families should be investigated carefully. Further research of this new family cancer syndrome may provide insight into the genetic basis of these diseases. (J P EDIATR 1996;128:220-4)

Autophagy, a well-conserved cellular self-eating process, has been shown to play a critical role in the pathophysiology of cancer. Previously, we reported that under normal O₂ conditions (21% O₂), the dual glucose metabolism inhibitor 2-deoxyglucose (2-DG) activates a cytoprotective autophagic response in cancer cells mainly through the induction of endoplasmic reticulum (ER) stress rather than ATP² reduction. However, the pathway(s) by which this occurs was unknown. Here, we find that ER stress induced by 2-DG as well as tunicamycin activates AMPK via Ca²⁺-CaMKKβ leading to stimulation of autophagy. These results suggest a new role for AMPK as a sensor of ER stress. In contrast, we find that although physiologic glucose starvation (GS) leads to ER stress which contributes to autophagy activation, it does so by a different mechanism. In addition to ER stress, GS also stimulates autophagy through lowering ATP and activating the canonical LKB1-AMPK energy sensing pathway as well as through increasing reactive oxygen species resulting in the activation of ERK. Furthermore, under hypoxia we observe that both 2-DG and GS inhibit rather than activate autophagy. This inhibition correlates with dramatically depleted ATP levels, and occurs through reduction of the PI3K III-Beclin1 complex for autophagy initiation, blockage of the conjugation of ATG12 to ATG5 for autophagosome expansion, as well as inhibition of the functional lysosomal compartment for autophagic degradation. Taken together, our data support a model where under normoxia therapeutic (2-DG) and physiologic (GS) glucose restriction differentially activate autophagy, while under hypoxia they similarly inhibit it.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children and the relapse rate in adult ALL patients is about 50%, highlighting the need for new therapeutic strategies. Data from our laboratory and others showed that ALL cells are sensitive to drugs that induce endoplasmic reticulum (ER) stress/unfolded protein response (UPR). In search for novel strategies to target the ER stress/UPR in ALL, we tested the efficacy of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924, pevo) in ALL. We found ALL cells exhibited significant in vitro and in vivo sensitivity to pevo-induced ER stress/UPR. Specifically, proteotoxic/ER stress was observed in pevo-treated ALL cells secondary to their inability to halt protein translation following pevo-induced activation of the mTOR pathway and concomitant de-phosphorylation of p-eIF2α (S51). In addition, aberrant activation of MEK/ERK has been correlated with resistance/relapse in pediatric ALL (Blood 2014; 124: 3420-3430). In our Bp- and T-ALL cell line models, we found consistent induction of p-ERK1/2 (T202/Y204) following pevo treatment, suggesting phosphorylation of ERK1/2 as a compensatory survival mechanism in response to pevo's cytotoxicity. Supporting this hypothesis, we observed significant in vitro synergy between the MEK inhibitor selumetinib (SEL) and pevo (CI = 0.017). On this basis, we tested the in vivo efficacy of pevo + SEL in NSG mice injected with NALM6 cells expressing the luciferase gene (NALM6/LUC). Engrafted NSG mice were treated with pevo (s.c., 66 mg/kg) and SEL (p.o., 50 mg/kg) twice daily on weekdays and once per day on weekends. Bioluminescence analysis of animals 21 days post ALL injection revealed significant reduction of tumor burden in mice treated with pevo alone or pevo + SEL (p Citation Format: Shuhua Zheng, Gilles M. Leclerc, Guy J. Leclerc, Joanna DeSalvo, Ronan T. Swords, Julio C. Barredo. Rebalancing of Bcl-2 family proteins mediate the vulnerability of pevonedistat-treated acute lymphoblastic leukemia cells towards MEK/ERK pathway inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4547.

10530Background: Acute Lymphoblastic Leukemia (ALL) remains a major cause of cancer-related deaths in children. We identified the AMP activated protein kinase (AMPK) as a potential target for ALL t...

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-d-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with d-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG–treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG–treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival. Mol Cancer Res; 10(7); 969–78. ©2012 AACR.