Search

The effective prophylaxis and treatment of central nervous system (CNS) involvement in acute lymphoblastic leukaemia (ALL) remains a significant clinical challenge. Developing novel and more effective CNS-directed therapies has been hampered, in part, by our limited understanding of the leukaemia niche in the CNS relative to the bone marrow. Accordingly, defining the molecular and cellular components critical for the establishment and maintenance of the CNS leukaemia niche may lead to new therapeutic opportunities. In prior work we showed that direct intercellular interactions between leukaemia and meningeal cells enhance leukaemia chemoresistance in the CNS. Herein, we show that the CXCR4/CXCL12 chemokine axis contributes to leukaemia-meningeal cell adhesion. Importantly, clinically tested CXCR4 antagonists, which are likely to cross the blood-brain and blood-cerebral spinal fluid barriers and penetrate the CNS, effectively disrupted leukaemia-meningeal cell adhesion. Moreover, by disrupting these intercellular interactions, CXCR4 antagonists attenuated leukaemia chemoresistance in leukaemia-meningeal cell co-culture experiments and enhanced the efficacy of cytarabine in targeting leukaemia cells in the meninges in vivo. This work identifies the CXCR4/CXCL12 axis as an important regulator of intercellular interactions within the CNS leukaemia niche and supports further testing of the therapeutic efficacy of CXCR4 antagonists in overcoming CNS niche-mediated chemoresistance.

The addition of molecularly targeted therapies to current chemotherapy regimens may improve acute lymphoblastic leukemia (ALL) outcomes and reduce acute and late toxicities. Checkpoint kinase 1 (CHK1) orchestrates cell cycle checkpoint control in the setting of DNA damage. CHK1 is expressed in both T- and B-ALL and represents a promising therapeutic target. Herein, we show that prexasertib, a targeted CHK1 inhibitor, exhibits significant single-agent efficacy in vivo using ALL patient-derived xenograft (PDX) models and synergizes in vitro with a nucleoside analog. These results support further clinical testing of prexasertib in ALL.

Background Obesity is a risk factor for many adulthood cancers, but its role in childhood, adolescent, and young adult (AYA) cancer is unknown. Childhood and AYA acute lymphoblastic leukemia (ALL) incidence and obesity prevalence have shown concurrent increases. We sought to identify whether obesity may be a risk factor for childhood and AYA ALL. Methods Characteristics from individuals with ALL, aged 2‐30 years, diagnosed 2004‐2017 and treated on Children's Oncology Group (COG) protocols with available pre‐treatment anthropometric data (N = 4726) were compared to National Health and Nutrition Examination Survey controls (COG AALL17D2). Body mass index (BMI) was defined using standard CDC definitions. Multivariate conditional logistic regression assessed associations between BMI and ALL with additional analyses stratified by sex and race/ethnicity. Results Among cases (72% high‐risk (HR) B‐ALL, 28% T‐ALL), 5% had underweight, 58% normal weight, 17% overweight, and 20% obesity. Underweight (OR 2.11, 95% CI 1.56‐2.85) and obesity (OR 1.32, 95% CI 1.15‐1.53) were associated with B‐ALL diagnosis. Specifically, obesity was associated with B‐ALL among males (OR 1.57, 95% CI 1.30‐1.91) and Hispanic children (OR 1.78, 95% CI 1.39‐2.29). Obesity was also associated with central nervous system (CNS) involvement. Conclusion Pre‐treatment obesity is associated with HR B‐ALL among males and Hispanics, as well as with CNS involvement, suggesting common physiology between obesity and leukemogenesis. An association between underweight and ALL was confirmed, likely due to cancer‐associated wasting. These results have important public health implications for obesity prevention and treatment in children and adolescents to reduce cancer risk., Deviations in body mass index (underweight and obesity) at the time of diagnosis were significantly associated with a diagnosis of pediatric high‐risk (HR) B‐cell acute lymphoblastic leukemia (B‐ALL) compared to normal weight. Specifically, obesity was associated with HR B‐ALL in males and Hispanics. Obesity was also significantly associated with ALL central nervous system (CNS) involvement.

Leukapheresis (LA) in pediatric leukemia is performed for leukostasis, a life-threatening emergency in the setting of extremely increased blast cell counts. The authors aimed to assess the epidemiology of pediatric leukemia who received LA. The authors reviewed US nationally representative admission records of patients less than 20 years of age in the Kids' Inpatient Database for the years 2000, 2003, 2006, 2009, 2012, and 2016. Incidence of new leukemia cases who underwent LA were calculated for the years 2009, 2012, and 2016. Cox and logistic regression analyses were performed to ascertain the risk factors for adverse outcomes. There were 526 admissions for pediatric patients with acute lymphoblastic leukemia (ALL) (n=328), acute myeloid leukemia (AML) (n=124), or chronic myeloid leukemia (CML) (n=74) who underwent LA over the study period. The incidence of leukemia cases that required LA was lower in 2016 than in 2009 or 2012 (1.4%, 2.2%, and 2.7%, respectively; P=0.001). In-hospital mortality was higher in AML than ALL (hzard ratio, 3.2; 95% confidence interval, 1.1-9.1). None with CML died during admission. This first population-based study of LA in pediatric leukemia showed a decreased utilization of LA over recent years. The higher inpatient mortality in AML, as compared with ALL or CML, warrant further investigations.

Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug.

Central nervous system (CNS) relapse is a common cause of treatment failure in patients with acute lymphoblastic leukaemia (ALL) despite current CNS-directed therapies that are also associated with significant short- and long-term toxicities. Herein, we showed that leukaemia cells exhibit decreased proliferation, elevated reactive oxygen species (ROS) and increased cell death in cerebral spinal fluid (CSF) both in vitro and in vivo. However, interactions between leukaemia and meningeal cells mitigated these adverse effects. This work expands our understanding of the pathophysiology of CNS leukaemia and suggests novel therapeutic approaches for more effectively targeting leukaemia cells in the CNS.

Protection from acute lymphoblastic leukemia (ALL) relapse in the central nervous system (CNS) is crucial to survival and quality of life for ALL patients. Current CNS-directed therapies cause significant toxicities and are only partially effective. Moreover, the impact of the CNS microenvironment on leukemia biology is poorly understood. Herein, we showed that leukemia cells associated with the meninges of xenotransplanted mice, or co-cultured with meningeal cells, exhibit enhanced chemoresistance due to effects on both apoptosis balance and quiescence. From a mechanistic standpoint, we identified that leukemia chemoresistance is primarily mediated by direct leukemia-meningeal cell interactions and overcome by detaching the leukemia cells from the meninges. Next, we used a co-culture adhesion assay to identify drugs that disrupted leukemia-meningeal adhesion. In addition to identifying several drugs that inhibit canonical cell adhesion targets we found that Me6TREN, a novel hematopoietic stem cell (HSC) mobilizing compound, also disrupts leukemia-meningeal adhesion in vitro and in vivo. Finally, Me6TREN enhanced the efficacy of cytarabine in treating CNS leukemia in xenotransplanted mice. This work demonstrates that the meninges exert a critical influence on leukemia chemoresistance, elucidates mechanisms of CNS relapse beyond the well-described role of the blood-brain barrier, and identifies novel therapeutic approaches for overcoming chemoresistance.

Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.

The limited delivery of chemotherapy agents to cancer cells and the nonspecific action of these agents are significant challenges in oncology. We have previously developed a customizable drug delivery and activation system in which a nucleic acid functionalized gold nanoparticle (Au-NP) delivers a drug that is selectively activated within a cancer cell by the presence of an mRNA unique to the cancer cell. The amount of drug released from sequestration to the Au-NP is determined by both the presence and the abundance of the cancer cell specific mRNA in a cell. We have now developed this technology for the potent, but difficult to deliver, topoisomerase I inhibitor SN-38. Herein, we demonstrate both the efficient delivery and selective release of SN-38 from gold nanoparticles in Ewing sarcoma cells with resulting efficacy in vitro and in vivo. These results provide further preclinical validation for this novel cancer therapy and may be extendable to other cancers that exhibit sensitivity to topoisomerase I inhibitors.

There are ˜3,000 children, as well an additional ˜7,000 adults, diagnosed with acute lymphoblastic leukaemia (ALL) each year in the USA. This makes ALL the most common cancer diagnosed in children. It represents ˜25% of paediatric cancer diagnoses. With current therapy, most patients achieve a complete remission and many are cured. However, the prognosis remains quite poor for the ˜15–20% of children who suffer a relapse of their ALL. Improved outcomes for these relapsed patients will require either more efficacious salvage therapies or improved initial therapy that prevents ALL relapse. Thus, understanding the mechanisms by which a small population of leukaemia cells can escape therapy and contribute to relapse often months or years later is critical for improving ALL outcomes. Herein, we will review emerging clinical and laboratory research that suggest quiescence, or dormancy, is an important cellular mechanism that enhances ALL chemo-resistance and persistence, and ultimately contributes to disease relapse. Furthermore, the mechanisms that regulate this balance between leukaemia quiescence and proliferation are beginning to be elucidated and will provide new knowledge about leukaemia biology. Finally, these observations support the need for and feasibility of therapeutically targeting these quiescent, chemo-resistant ALL cells by either exploiting metabolic or signalling pathway vulnerabilities unique to quiescent cells, or by causing the release of ALL cells from the protective niche(s) that triggers and maintains ALL quiescence.

Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV+ DLBCL) in pediatrics most commonly occurs as an iatrogenic immunodeficiency-associated lymphoproliferative disease. We report an 18-year-old female individual with refractory systemic juvenile idiopathic arthritis, treated with multiple immunosuppressive agents, who was diagnosed with stage III, EBV+ DLBCL. The patient achieved sustained complete remission after 4 weekly doses of rituximab monotherapy and reduction of immunosuppression. This case suggests that a post-transplant lymphoproliferative disease-like treatment approach can be a safe and effective therapy in a nontransplant, yet severely immunosuppressed, patient with EBV+ DLBCL.

Central nervous system (CNS) relapse is a common cause of treatment failure in patients with acute lymphoblastic leukemia (ALL) despite current CNS-directed therapies that are also associated with significant short and long-term toxicities. Herein, we showed that leukemia cells exhibit decreased proliferation, elevated reactive oxygen species (ROS), and increased cell death in CSF both in vitro and in vivo. However, interactions between leukemia and meningeal cells mitigated these adverse effects. This work expands our understanding of the pathophysiology of CNS leukemia and suggests novel therapeutic approaches for more effectively targeting leukemia cells in the CNS.

Cytokines secreted by bone marrow stromal cells protect c-KIT mutant AML cells from c-KIT inhibitor-induced apoptosis

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and a leading cause of cancer death in children.[1] The central nervous system (CNS) is a frequent site of leukemia involvemen...

A pair of synthetic approaches to linear dasatinib–DNA conjugates via click chemistry are described. The first approach involves the reaction of excess azido dasatinib derivative with 5′-(5-hexynyl)-tagged DNAs, and the second involves the reaction of excess alkynyl-linked dasatinib with 5′-azido-tagged DNA. The second approach using alkynyl-derived dasatinib and 5′-azido-tagged DNA yielded the corresponding dasatinib-DNA conjugates in higher yield (47% versus 10–33% for the first approach). Studies have shown these linear dasatinib–DNA conjugates-derived gold nanoparticles exhibit efficacy against leukemia cancer cells with reduced toxicity toward normal cells compared to that of free dasatinib.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While significant progress has been made in the therapy of leukemia, several obstacles still hinder cure. Central nervous system (CNS) relapse is a major cause of treatment failure among patients with ALL and current CNS-directed therapies are also associated with significant morbidities including neurocognitive deficits, endocrinopathies, and secondary malignancies. Consequently, novel CNS-directed leukemia therapies are needed to improve long-term outcomes in ALL while decreasing treatment-related morbidity. We developed in vitro and in vivo models of CNS leukemia in order to identify the unique characteristics of the CNS microenvironment that create a sanctuary site for leukemia cells. We transplanted multiple human ALL cell lines into immune-compromised mice (NSG). Mice were not irradiated or conditioned prior to transplantation to avoid perturbing leukemia niches. Using this xenotransplant model, we then identified the meninges as the predominant CNS site that harbors leukemia cells both before and after treatment with systemic cytarabine. This anatomic distribution of CNS leukemia agrees with other murine studies and also recapitulates the histopathology of human CNS leukemia. Having demonstrated that the meninges provide a unique niche for leukemia, we then developed ex vivo co-culture approaches to focus more specifically on the effects of the meninges on leukemia biology. To more accurately model the leukemia-meningeal niche in co-culture we substituted tissue culture media for cerebral spinal fluid (CSF). However, given the unique composition and concentration of many substrates in CSF relative to serum or media we first characterized the effects of CSF on leukemia cells. We found that leukemia cells in CSF have limited survival even when replenished daily with fresh CSF. We hypothesized this decrease in viability may be secondary to elevated reactive oxygen species (ROS) given the lower levels of redox proteins in CSF. Accordingly, we found leukemia cells in CSF showed a higher level of ROS compared to the leukemia cells in regular media. Addition of N-acetyl-cysteine, a ROS scavenger, to CSF decreased ROS levels and cell death in leukemia cells. Moreover, leukemia cells co-cultured with meningeal cells in CSF showed decreased ROS levels compared to leukemia cells growing in suspension in CSF. We found the effect of the meninges on leukemia biology extends beyond ROS regulation. Leukemia cells co-cultured with meningeal cells were also significantly more resistant to chemotherapy-induced apoptosis through effects on apoptosis balance and enhanced quiescence. These effects were reversed when the leukemia cells were removed from the meninges and placed back into suspension. Moreover, leukemia cells cultured in meningeal conditioned media also exhibited mild chemoresistance, indicating a role for a soluble factor(s) secreted by meningeal cells. Using proteomic approaches we then identified candidate soluble factor(s) secreted by meningeal cells that may contribute to leukemia chemoresistance. These results show that meningeal cells influence key aspects of leukemia biology, including ROS regulation and chemoresistance, and that the pathophysiology of CNS leukemia is not only related to the ability of leukemia cells and chemotherapy to access the restricted CNS environment. Finally we are leveraging this knowledge of the CNS leukemia niche into the development of novel CNS-directed therapies that modulate ROS levels, target candidate soluble factors, or that disrupt the interactions between leukemia and meningeal cells. Disclosures No relevant conflicts of interest to declare.

Central nervous system (CNS) relapse is a leading cause of treatment failure in acute lymphoblastic leukemia (ALL). We have shown that the meninges provide a unique leukemia niche in the CNS that enhances leukemia chemoresistance through effects on apoptosis and quiescence. We now describe our work to leverage this new knowledge of the CNS leukemia niche into novel CNS-directed leukemia therapies that 1) target vulnerabilities unique to leukemia cells in the meninges, 2) inhibit the meningeal pathways that contribute to leukemia chemoresistance, and 3) disrupt the interactions between leukemia and meningeal cells. First, we focused on identifying and targeting vulnerabilities unique to leukemia cells in the meninges. We used a leukemia-meningeal co-culture system and tested the ability of drugs targeting pathways important for leukemia biology to overcome meningeal-mediated leukemia resistance to cytarabine and methotrexate. Intriguingly, we found that pre-treatment of leukemia cells with ruxolitinib, a JAK1/2 inhibitor, prior to co-culture with primary meningeal cells diminished leukemia chemoresistance. Ruxolitinib had no effect on the chemosensitivity of leukemia cells in suspension. Other JAK1/2 inhibitors, such as CHZ868, yielded comparable results and we are currently testing the ability of JAK2 knockdown by shRNA in leukemia cells to overcome chemoresistance. As a potential explanation for the mechanism by which ruxolitinib overcomes meningeal-mediated leukemia resistance, we found that ruxolitinib decreases leukemia quiescence and enhances proliferation in co-culture, as measured by FxCycle and Ki67 staining. Finally, preliminary experiments in xenotransplanted mice showed that ruxolitinib significantly enhanced the efficacy of cytarabine in treating leukemia in the meninges. Second, we also identified and therapeutically targeted meningeal signaling pathways that contribute to leukemia chemoresistance. We used reverse phase protein arrays (RPPA; MD Anderson) to assess the effects of co-culture on protein expression and phosphorylation in primary meningeal cells. RPPA determines the levels of >300 proteins involved in multiple signaling pathways. Amongst other findings, we identified activation of the AKT pathway in primary meningeal cells in the presence of leukemia cells. Furthermore, pre-treatment of primary meningeal cells with INK128, an inhibitor that acts downstream of AKT and targets mTOR, prior to co-culture with leukemia cells diminished leukemia chemoresistance. Third, we tested whether disrupting meningeal-leukemia adhesion overcomes leukemia chemoresistance. Leukemia cells were dissociated from meningeal cells with trypsin and manual pipetting after co-culture for 48-72 hours. Leukemia cells were then purified with magnetic beads and placed back in suspension prior to further characterization and drug testing. We found leukemia cells placed back into suspension after co-culture with meningeal cells, or isolated from the meninges of mice, reverted back to baseline cell cycle, quiescence, and apoptosis balance characteristics. Moreover, leukemia cells removed from co-culture exhibited similar sensitivity to methotrexate and cytarabine as leukemia cells in suspension. These results suggest that drugs or biologic agents that disrupt adhesion between leukemia and meningeal cells may restore leukemia chemosensitivity in the CNS niche. Accordingly, we next identified several cell adhesion inhibitors that effectively disrupted leukemia-meningeal adhesion in co-culture. The more promising agents are currently being characterized more extensively in co-culture and tested in combination with chemotherapy in xenotransplanted mice. In summary, in order to address the need for more efficacious and less toxic therapies for CNS leukemia, we have identified mechanisms by which the meninges enhance leukemia chemoresistance. We are now testing novel strategies for overcoming leukemia chemoresistance that by targeting different components of the CNS leukemia niche (leukemia cells, meningeal cells, or the interactions between the two cell types) may be complementary or even synergistic. Disclosures No relevant conflicts of interest to declare.

// Nathan P. Gossai 1, * , Jordan A. Naumann 1, * , Nan-Sheng Li 4 , Edward A. Zamora 1 , David J. Gordon 3 , Joseph A. Piccirilli 4, 5 , Peter M. Gordon 1, 2 1 Department of Pediatrics, Division of Pediatric Hematology and Oncology, University of Minnesota, Minneapolis, MN, USA 2 University of Minnesota Masonic Cancer Center, Minneapolis, MN, USA 3 Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Iowa, Iowa City, IA, USA 4 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA 5 Department of Chemistry, University of Chicago, Chicago, IL, USA * These authors have contributed equally to this work Correspondence to: Peter M. Gordon, email: gord0047@umn.edu Keywords: gold nanoparticles, drug delivery, leukemia, molecularly targeted therapy, anti-sense Received: February 08, 2016 Accepted: May 01, 2016 Published: May 18, 2016 ABSTRACT We describe a customizable approach to cancer therapy in which a gold nanoparticle (Au-NP) delivers a drug that is selectively activated within the cancer cell by the presence of an mRNA unique to the cancer cell. Fundamental to this approach is the observation that the amount of drug released from the Au-NP is proportional to both the presence and abundance of the cancer cell specific mRNA in a cell. As proof-of-principle, we demonstrate both the efficient delivery and selective release of the multi-kinase inhibitor dasatinib from Au-NPs in leukemia cells with resulting efficacy in vitro and in vivo . Furthermore, these Au-NPs reduce toxicity against hematopoietic stem cells and T-cells. This approach has the potential to improve the therapeutic efficacy of a drug and minimize toxicity while being highly customizable with respect to both the cancer cell specific mRNAs targeted and drugs activated.

SummaryGroup II introns are mobile genetic elements that have been implicated as agents of genetic diversity, and serve as important model systems for investigating RNA catalysis and pre-mRNA splicing. In the absence of an atomic-resolution structure of the intron, detailed understanding of its catalytic mechanism has remained elusive. Previous identification of a divalent metal ion stabilizing the leaving group in both splicing steps suggested that the group II intron may employ a “two-metal ion” mechanism, a catalytic strategy used by a number of protein phosphoester transfer enzymes. Using metal rescue experiments, we now reveal the presence of a second metal ion required for nucleophile activation in the exon-ligation step of group II intron splicing. Coupled with biochemical and structural evidence of at least two metal ions at the group I intron reaction center, these results suggest a mechanistic paradigm for describing catalysis by large ribozymes.

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal disorders of the haematopoietic stem cell and primarily involve cells of the myeloid lineage. Using cDNA microarrays comprising 6000 human genes, we studied the gene expression profiles in the neutrophils of 21 MDS patients, seven of which had the 5q- syndrome, and two acute myeloid leukaemia (AML) patients when compared with the neutrophils from pooled healthy controls. Data analysis showed a high level of heterogeneity of gene expression between MDS patients, most probably reflecting the underlying karyotypic and genetic heterogeneity. Nevertheless, several genes were commonly up or down-regulated in MDS. The most up-regulated genes included RAB20, ARG1, ZNF183 and ACPL. The RAB20 gene is a member of the Ras gene superfamily and ARG1 promotes cellular proliferation. The most down-regulated genes include COX2, CD18, FOS and IL7R. COX2 is anti-apoptotic and promotes cell survival. Many genes were identified that are differentially expressed in the different MDS subtypes and AML. A subset of genes was able to discriminate patients with the 5q- syndrome from patients with refractory anaemia and a normal karyotype. The microarray expression results for several genes were confirmed by real-time quantitative polymerase chain reaction. The MDS-specific expression changes identified are likely to be biologically important in the pathophysiology of this disorder.

Group II introns require numerous divalent metal ions for folding and catalysis. However, because little information about individual metal ions exists, elucidating their ligands, functional roles and relationships to each other remains challenging. Here we provide evidence that an essential motif at the catalytic center of the group II intron, the AGC triad within domain 5 (D5), provides a ligand for a crucial metal ion. Sulfur substitution of the pro-Sp oxygen of the adenosine strongly disrupts D5 binding to a substrate consisting of an exon and domains 1–3 of the intron (exD123). Cd2+ rescues this effect by enabling the sulfur-modified D5 to bind to exD123 with wild type affinity and catalyze 5′-splice site cleavage. This switch in metal specificity implies that a metal ion interacts with D5 to mediate packing interactions with D123. This new D5 metal ion rescues the disruption of D5 binding and catalysis with a thermodynamic signature different from that of the metal ion that stabilizes the leaving group during the first step of splicing, suggesting the existence of two distinct metal ions.

The ai5gamma group II intron from yeast excises itself from precursor transcripts in the absence of proteins. When a shortened form of the intron containing all but the 3'-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3' splice site oligonucleotide, a nucleotidyl transfer reaction occurs that mimics the second step of splicing. As this tripartite reaction provides a means to identify important functional groups in 3' splice site recognition and catalysis, we establish here a minimal kinetic framework and demonstrate that the chemical step is rate-limiting. We use this framework to characterize the metal ion specificity switch observed previously upon sulfur substitution of the 3'-oxygen leaving group and to elucidate by atomic mutagenesis the role of the neighboring 2'-OH in catalysis. The results suggest that both the 3'-oxygen leaving group and the neighboring 2'-OH are important ligands for metal ions in the transition state but not in the ground state and that the 2'-OH may play an additional role in transition state stabilization by donating a hydrogen bond. Metal specificity switch experiments combined with quantitative analysis show that the Mn(2+) that interacts with the leaving group binds to the ribozyme with the same affinity as the metal ion that interacts with the neighboring 2'-OH, raising the possibility that a single metal ion mediates interactions with the 2'- and 3'-oxygen atoms at the 3' splice site.

Central nervous system (CNS) relapse is a significant cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). Isolated CNS relapse occurs in ~3-8% of children with leukemia and accounts for 30-40% of initial relapses in some clinical trials. In addition, CNS-directed therapies are associated with seizures, secondary neoplasms, encephalopathy, and long-term endocrine, developmental, neurovascular, and cognitive deficits. While many studies have demonstrated that interactions between leukemia cells and components of the bone marrow microenvironment influence leukemia development, maintenance, and chemo-resistance, the role of the CNS microenvironment in leukemia is less well studied. To investigate the influence of the CNS niche in leukemia, we asked whether the CNS niche could impart unique and functionally important gene expression changes in leukemia cells. We transplanted NALM-6 human, pre-B leukemia cells into NSG mice without prior irradiation to avoid perturbing the bone marrow and CNS niches. After systemic leukemia development the mice were euthanized and leukemia cells were isolated from the bone marrow and CNS microenvironments. Gene expression profiling of ~700 cancer-associated genes (NanoString® Technology) identified 36 leukemia genes differentially expressed (30 up-regulated and 6 down-regulated; fold change ≥ 2 and FDR We elected to further examine the gene PBX1 as it is a transcription factor with known roles in hematopoiesis, leukemia, and cancer biology. PBX1 contributes to Evi-1 mediated leukemia development in a murine model of leukemia and is a partner with TCF3 in the t(1;19) translocation that occurs in ~5% of pre-B ALL. Interestingly, this translocation is also associated with an increased risk for CNS relapse. We confirmed PBX1 mRNA up-regulation in leukemia cells either isolated from the CNS or in leukemia cells co-cultured with CNS-derived, murine choroid plexus cells by quantitative PCR. Supporting the generalizability of these results, additional human B-cell leukemia lines SEM and REH also exhibited PBX1 mRNA up-regulation in leukemia cells isolated from the murine CNS niche. Western blots of NALM-6 and SEM leukemia cells isolated from the mouse CNS as well as from leukemia cells co-cultured ex vivo with choroid plexus cells also showed PBX1 up-regulation at the protein level. Finally, culture of leukemia cells in either choroid plexus cell conditioned media or human cerebral spinal fluid (CSF) had minimal effects on PBX1 protein levels, suggesting that direct cell contact, rather than a soluble factor(s), contributes to PBX1 up-regulation in leukemia cells. Following confirmation of PBX1 up-regulation in leukemia cells within the CNS microenvironment, we next ectopically expressed PBX1, or GFP as a control, in leukemia cells to identify functional consequences of PBX1 expression in leukemia cells. Leukemia cells expressing PBX1 exhibited decreased sensitivity to cytarabine relative to control cells as measured by both proliferation and apoptosis assays. Furthermore, shRNA targeting PBX1, but not control shRNA, prevented PBX1 up-regulation in leukemia cells when co-cultured with choroid plexus cells and modestly, but significantly, attenuated the ability of choroid plexus cells to protect leukemia cells from cyatabine-induced apoptosis. Finally, although PBX1 had no effect on leukemia proliferation, it caused enhanced colony-forming ability in semi-solid media, consistent with increased leukemia self-renewal properties. Together these results suggest PBX1 up-regulation in leukemia cells may contribute to both leukemia self-renewal properties and chemo-resistance in the CNS niche. In summary, we believe this work illustrates the unique and functionally important effect that the CNS microenvironment has on leukemia cells. More comprehensive analyses of the leukemia transcriptome, genome, and proteome in the CNS niche will build upon this foundation and provide a more detailed understanding of the role of the CNS niche in leukemia biology. Finally, this approach may identify targetable CNS niche-mediated mechanisms of leukemia chemo-resistance and self-renewal. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Novel therapies are needed for acute myeloid leukemia as only ~60% of children are cured despite maximally intensive cytotoxic chemotherapy. Functionalized gold nanoparticles (AuNP) are utilized for many biomedical applications and represent a potentially novel therapeutic approach in leukemia. We enhanced this technology by developing an AuNP system that selectively releases molecularly targeted drugs in leukemia cells. METHODS: AuNPs were functionalized with short, double-stranded oligonucleotides with sequence complementarity to genes overexpressed in or unique to a leukemia cell (e.g. survivin or AML/ETO). Only the anti-sense oligonucleotide is covalently bound to the nanoparticle via a thiol linker. Thus, after entering leukemia cells, the endogenous targeted oncogene mRNA can bind to its complementary sequence on the AuNP and displace the non-covalently bound oligonucleotide which, in our system, is conjugated to the multi-tyrosine kinase inhibitor dasatinib. As a binary reaction, the amount of dasatinib-conjugated oligonucleotide released from the nanoparticle is directly proportional to both the presence and abundance of the complementary mRNA present in a cell. We evaluated AuNP uptake into multiple AML cell lines, as well as normal hematopoietic cells. The effect of dasatinib-AuNPs on dasatinib-sensitive leukemia cell lines was evaluated using proliferation assays, annexin V staining, and cell colony assays. Toxicity in T-cells and CD34+ cells was assessed with T-cell activation and p-SRC assays, respectively. RESULTS: Conjugation of dasatinib to an oligonucleotide complementary to a region of the survivin gene did not perturb its ability to inhibit SRC and c-KIT kinases in vitro. Leukemia cells demonstrate highly efficient AuNP uptake when cultured alone or with up to a 100-1000 fold excess of normal bone marrow cells. Treatment of K562 leukemia cells, containing a BCR/ABL translocation and high levels of survivin mRNA, with dasatinib-AuNPs resulted in dose-dependent p-SRC and p-CRKL inhibition. Furthermore, K562 cells also showed significantly impaired proliferation, increased apoptosis, and formed fewer colonies in methylcellulose. Conversely, normal T-cells and CD34+ cells, which express less survivin than leukemia cells, were significantly less affected by dasatinib-AuNPs than dasatinib alone as measured by T-cell activation assays and p-SRC levels, respectively. CONCLUSIONS: This method of using functionalized AuNPs to deliver and activate dasatinib in leukemia cells augments drug efficacy while minimizing toxicity and thus represents a novel therapeutic strategy for AML. Ongoing work is underway to characterize the mechanism of preferential AuNP uptake in leukemia cells and assessing in vivo activity using a murine xenotransplantation model of leukemia. Disclosures No relevant conflicts of interest to declare.

Mechanistic analyses of nuclear pre-mRNA splicing by the spliceosome and group II intron self-splicing provide insight into both the catalytic strategies of splicing and the evolutionary relationships between the different splicing systems. We previously showed that 39-sulfur substitution at the 39 splice site of a nuclear pre-mRNA has no effect on splicing. We now report that 39-sulfur substitution at the 39 splice site of a nuclear pre-mRNA causes a switch in metal specificity when the second step of splicing is monitored using a bimolecular exon-ligation assay. This suggests that the spliceosome uses a catalytic metal ion to stabilize the 39-oxyanion leaving group during the second step of splicing, as shown previously for the first step. The lack of a metal-specificity switch under cis splicing conditions indicates that a rate-limiting conformational change between the two steps of splicing may mask the subsequent chemical step and the metal-specificity switch. As the group II intron, a true ribozyme, uses identical catalytic strategies for splicing, our results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns.

The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor. The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question. Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related. Here we show that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing. In contrast, 3'-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing. Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction. These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing. Furthermore, because 3'-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems.

A case of emphysematous pyelonephritis is presented in which computerized tomography (CT) both confirmed the diagnosis and demonstrated extension of the gas to the region of the adrenal glands and inferior cava. The etiology and pathogenesis of emphysematous pyelonephritis is discussed. Radiologic evaluation of this disease should be prompt and noninvasive. Plain films establish the diagnosis, isotope scanning assesses function of the opposite kidney, and CT demonstrates the full extent of the pathological process and confirms the diagnosis. Aggressive treatment is recommended.

The 2′-hydroxyl group contributes inextricably to the functional behavior of many RNA molecules, fulfilling numerous essential chemical roles. To assess how hydroxyl groups impart functional behavior to RNA, we developed a series of experimental strategies using an array of nucleoside analogs. These strategies provide the means to investigate whether a hydroxyl group influences function directly (via hydrogen bonding or metal ion coordination), indirectly (via space-filling capacity, inductive effects, and sugar conformation), or through interactions with solvent. The nucleoside analogs span a broad range of chemical diversity, such that quantitative structure activity relationships (QSAR) now become possible in the exploration of RNA biology. We employed these strategies to investigate the spliced exons reopening (SER) reaction of the group II intron. Our results suggest that the cleavage site 2′-hydroxyl may mediate an interaction with a water molecule.