Peter M. Gordon

Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis

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.

Role for the Proapoptotic Factor BIM in Mediating Imatinib-induced Apoptosis in a c-KIT-dependent Gastrointestinal Stromal Tumor Cell Line

The c-KIT receptor tyrosine kinase is constitutively activated and oncogenic in the majority of gastrointestinal stromal tumors. The identification of selective inhibitors of c-KIT, such as imatinib, has provided a novel therapeutic approach in the treatment of this chemotherapy refractory tumor. However, despite the clinical importance of these findings and the potential it provides as a model system for understanding targeted therapy, this approach has not yielded curative outcomes in most patients, and the biochemical pathways connecting c-KIT inhibition to cell death are not completely understood. Here, we show that inhibition of c-KIT with imatinib in gastrointestinal stromal tumors (GISTs) triggered the up-regulation of the proapoptotic protein BIM via both transcriptional and post-translational mechanisms. The inhibition of c-KIT by imatinib increased levels of the dephosphorylated and deubiquitinated form of BIM as well as triggered the accumulation of the transcription factor FOXO3a on the BIM promoter to activate transcription of BIM mRNA. Furthermore, using RNA interference directed against BIM, we demonstrated that BIM knockdown attenuated the effects of imatinib, suggesting that BIM functionally contributes to imatinib-induced apoptosis in GIST. The identification and characterization of the pathways that mediate imatinib-induced cell death in GIST provide for a better understanding of targeted therapy and may facilitate the development of new therapeutic approaches to further exploit these pathways.

Drug conjugated nanoparticles activated by cancer cell specific mRNA

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.

The central nervous system microenvironment influences the leukemia transcriptome and enhances leukemia chemo-resistance

Central nervous system (CNS) relapse is a principal cause of treatment failure among patients with acute lymphoblastic leukemia (ALL). Isolated CNS relapse occurs in approximately 3%–8% of children with leukemia and accounts for 30%–40% of initial relapses in some clinical trials.[1][1]

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

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

In vitro model of leukemia cell migration across the blood-cerebrospinal fluid barrier.

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 involvement and a principal cause of treatment failure among patients with ALL.[2] In addition, CNS-directed therapies are associated with significant short and long-term toxicities including seizures, secondary neoplasms, encephalopathy, and late endocrine, developmental, neurovascular, and cognitive deficits.[3] Accordingly, there is a significant need for more efficacious and less toxic therapies for treating CNS leukemia. For CNS leukemia to occur, leukemia cells must migrate from the bone marrow, in which leukemia develops, to the CNS. This is an incompletely understood and potentially complex process given that the CNS is an immunologically privileged site with restricted access for both benign and malignant immune cells.[4] Potential sites of leukemia cell entry into the CNS include the blood–brain barrier (BBB), meningeal vasculature, and the blood–cerebral spinal fluid barrier (BCSFB).[4,5] Both postmortem CNS examinations of ALL patients and murine leukemia xenotransplantation studies suggest that leukemia cells transit the BCSFB barrier that is composed of the choroid plexus (CP).[6,7] The CP extends from the ependymal cells that line the ventricles and consists of a layer of cuboidal, polarized epithelial cells surrounding capillaries and stromal elements. Within the CP, the vascular endothelium is fenestrated and, as a result, the BCSFB barrier occurs at the CP epithelium, which contains tight junctions.[5] Studying leukemia cell migration through the BCSFB in vivo is challenging as a result of the poor accessibility of the CP within the ventricles. As a result, the mechanism by which leukemia cells transit from the systemic circulation to the CNS is poorly understood but important for understanding and treating CNS leukemia. To address this shortcoming, we have modified an in vitro model of the BCSFB using immortalized murine CP epithelial cells (Z310) for use in investigating leukemia cell transit across the CP epithelium.[8] Z310 cells express major CP tight junction proteins, form tight barriers, and have been used extensively for pharmacological and toxicological studies of transport at the BCSFB.[9] In our assay, Z310 CP cells were seeded on the upperside of a 8 lM ThinCert Cell Culture Inserts (Greiner Bio-One, Monroe, NC) and cultured in regular media for 24–48 h until confluent (Figure 1(A)). Several approaches were taken to ensure Z310 cell confluency including demonstrating the cells (1) were impermeable to media flow-through, (2) exhibited a stable Trans Epithelial Electric Resistance (TEER) value across the cell layer, and (3) formed a confluent layer with no gaps when examined by microscopy (Supplementary Figure 1(A–C)). Only inserts that satisfied these criteria were used for further studies. While these experiments confirm the barrier function of the CP epithelial cells, a limitation of this assay design is that it overlooks the extracellular matrix and inflammatory cells, including macrophages and dendritic cells, present in the CP in vivo.[5] Both these CP components may influence CP function and leukemia cell migration. After the Z310 cells were confluent, the insert was inverted and 2.5 10 dye-labeled Jurkat, CEM, or primary T-cell leukemia cells (T-ALL) were applied to the underside of the membrane that has been treated with retronectin to enhance leukemia cell adhesion. After allowing the leukemia cells to adhere for 20min, the insert was placed back upright into the well. This configuration correctly models the in vivo migration of leukemia cells from the blood to the CSF, as the leukemia cells must traverse from the basolateral to the apical side of the Z310 cells. After 24 h, the media from the upper well of the insert was removed and the number of leukemia cells assessed by flow cytometry. Dye-labeled leukemia cells allowed us to discriminate between leukemia cells

SN-38 Conjugated Gold Nanoparticles Activated by Ewing Sarcoma Specific mRNAs Exhibit In Vitro and In Vivo Efficacy

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.

The Role of the Central Nervous System Microenvironment in Pediatric Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. While survival rates for ALL have improved, central nervous system (CNS) relapse remains a significant cause of treatment failure and treatment-related morbidity. Accordingly, there is a need to identify more efficacious and less toxic CNS-directed leukemia therapies. Extensive research has demonstrated a critical role of the bone marrow (BM) microenvironment in leukemia development, maintenance, and chemoresistance. Moreover, therapies to disrupt mechanisms of BM microenvironment-mediated leukemia survival and chemoresistance represent new, promising approaches to cancer therapy. However, in direct contrast to the extensive knowledge of the BM microenvironment, the unique attributes of the CNS microenvironment that serve to make it a leukemia reservoir are not yet elucidated. Recent work has begun to define both the mechanisms by which leukemia cells migrate into the CNS and how components of the CNS influence leukemia biology to enhance survival, chemoresistance, and ultimately relapse. In addition to providing new insight into CNS relapse and leukemia biology, this area of investigation will potentially identify targetable mechanisms of leukemia chemoresistance and self-renewal unique to the CNS environment that will enhance both the durability and quality of the cure for ALL patients.