Instructor, Pediatrics - Hematology & Oncology
BS, University of Illinois, Biology, Electrical Engineering (1995)
MD, PhD, Washington University, Molecular Cell Biology (2002)
Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.
View details for DOI 10.1016/j.chembiol.2013.09.014
View details for Web of Science ID 000328434700008
View details for PubMedID 24183972
Disseminated infection due to nontuberculous Mycobacterium (NTM) species is rare in pediatrics. Here we report 6 infections affecting 5 patients at a single institution in an immunocompromised population of pediatric oncology and stem cell transplant recipients. The patients presented within a 1-year period with catheter-associated bacteremia. New pulmonary nodules were noted in 4 of the 5 patients. All of the infections were due to rapidly growing NTM. Patients were successfully treated with removal of the infected catheter and combination antibiotic therapy. There are currently no consensus guidelines for treatment of NTM infections in this population, and a therapeutic approach is presented here.
View details for Web of Science ID 000268815000006
View details for PubMedID 19641470
A 7-year-old boy presented with fulminant hepatic failure requiring liver transplant. Serologic testing ruled out infectious and autoimmune causes. During transplant surgery he was found to have enlarged periportal lymph nodes that were biopsied. Nodular lymphocyte-predominant Hodgkin lymphoma was diagnosed based on histologic examination of the lymph node and liver. The L&H cells within the lymph node were positive for CD20 whereas those within the liver were not, although they were positive for other B-cell markers. After extensive work-up, the cause of liver failure could only be attributed to the involvement by lymphoma. In addition, B-cell clonality was established among the neoplastic cells with the same clone detected in all sampled tissues. Hodgkin lymphoma as a cause of hepatic failure is rare and has not been previously reported in a pediatric patient.
View details for Web of Science ID 000253788700016
View details for PubMedID 18227720
The proto-oncogene BCL-2 was discovered with the cloning of the t(14;18) chromosomal translocation responsible for human follicular lymphoma. Since then other members of the Bcl-2 family of cell death regulators have been identified and their roles in cell death, normal lymphoid development, and lymphoid neoplasia have been characterized. Bcl-2 family members are important in tumor initiation, progression, and response to chemotherapy, and altered expression levels of various members serve as prognostic markers in many lymphoid malignancies. There are promising cancer therapeutics now targeted at members of the Bcl-2 family.
View details for DOI 10.1532/IJH97.04110
View details for Web of Science ID 000224672700001
View details for PubMedID 15540893
Critical issues in apoptosis include the importance of caspases versus organelle dysfunction, dominance of anti- versus proapoptotic BCL-2 members, and whether commitment occurs upstream or downstream of mitochondria. Here, we show cells deficient for the downstream effectors Apaf-1, Caspase-9, or Caspase-3 display only transient protection from "BH3 domain-only" molecules and die a caspase-independent death by mitochondrial dysfunction. Cells with an upstream defect, lacking "multidomain" BAX, BAK demonstrate long-term resistance to all BH3 domain-only members, including BAD, BIM, and NOXA. Comparison of wild-type versus mutant BCL-2, BCL-X(L) indicates these antiapoptotics sequester BH3 domain-only molecules in stable mitochondrial complexes, preventing the activation of BAX, BAK. Thus, in mammals, BH3 domain-only molecules activate multidomain proapoptotic members to trigger a mitochondrial pathway, which both releases cytochrome c to activate caspases and initiates caspase-independent mitochondrial dysfunction.
View details for Web of Science ID 000171251500024
View details for PubMedID 11583631
Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.
View details for Web of Science ID 000168478300051
View details for PubMedID 11326099
Multiple apoptotic pathways release cytochrome c from the mitochondrial intermembrane space, resulting in the activation of downstream caspases. In vivo activation of Fas (CD95) resulted in increased permeability of the mitochondrial outer membrane and depletion of cytochrome c stores. Serial measurements of oxygen consumption, NADH redox state and membrane potential revealed a loss of respiratory state transitions. This tBID-induced respiratory failure did not require any caspase activity. At early time points, re-addition of exogenous cytochrome c markedly restored respiratory functions. Over time, however, mitochondria showed increasing irreversible respiratory dysfunction as well as diminished calcium buffering. Electron microscopy and tomographic reconstruction revealed asymmetric mitochondria with blebs of herniated matrix, distended inner membrane and partial loss of cristae structure. Thus, apoptogenic redistribution of cytochrome c is responsible for a distinct program of mitochondrial respiratory dysfunction, in addition to the activation of downstream caspases.
View details for Web of Science ID 000167087200004
View details for PubMedID 11179211
Many apoptotic molecules relocate subcellularly in cells undergoing apoptosis. The pro-apoptotic protein BID underwent posttranslational (rather than classic cotranslational) N-myristoylation when cleavage by caspase 8 caused exposure of a glycine residue. N-myristoylation enabled the targeting of a complex of p7 and myristoylated p15 fragments of BID to artificial membranes bearing the lipid composition of mitochondria, as well as to intact mitochondria. This post-proteolytic N-myristoylation serves as an activating switch, enhancing BID-induced release of cytochrome c and cell death.
View details for Web of Science ID 000165632400042
View details for PubMedID 11099414
We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c approximately 20A. A definable pore progressed from approximately 11A consisting of two BAX molecules to a approximately 22A pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c. Cell Death and Differentiation (2000) 7, 1166 - 1173
View details for Web of Science ID 000166010000004
View details for PubMedID 11175253
TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.
View details for Web of Science ID 000088871600008
View details for PubMedID 10950869
Progression of cells through the G1 phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G1 cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G1 phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G1.
View details for Web of Science ID 000080712700015
View details for PubMedID 10363976
"BH3 domain only" members of the BCL-2 family including the pro-apoptotic molecule BID represent candidates to connect with proximal signal transduction. Tumor necrosis factor alpha (TNFalpha) treatment induced a caspase-mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites to yield a major p15 and minor p13 and p11 fragments. p15 BID translocates to mitochondria as an integral membrane protein. p15 BID within cytosol targeted normal mitochondria and released cytochrome c. Immunodepletion of p15 BID prevents cytochrome c release. In vivo, anti-Fas Ab results in the appearance of p15 BID in the cytosol of hepatocytes which translocates to mitochondria where it releases cytochrome c. Addition of activated caspase-8 to normal cytosol generates p15 BID which is also required in this system for release of cytochrome c. In the presence of BCL-XL/BCL-2, TNFalpha still induced BID cleavage and p15 BID became an integral mitochondrial membrane protein. However, BCL-XL/BCL-2 prevented the release of cytochrome c, yet other aspects of mitochondrial dysfunction still transpired and cells died nonetheless. Thus, while BID appears to be required for the release of cytochrome c in the TNF death pathway, the release of cytochrome c may not be required for cell death.
View details for Web of Science ID 000077968500079
View details for PubMedID 9873064
Expression of the pro-apoptotic molecule BAX has been shown to induce cell death. While BAX forms both homo- and heterodimers, questions remain concerning its native conformation in vivo and which moiety is functionally active. Here we demonstrate that a physiologic death stimulus, the withdrawal of interleukin-3 (IL-3), resulted in the translocation of monomeric BAX from the cytosol to the mitochondria where it could be cross-linked as a BAX homodimer. In contrast, cells protected by BCL-2 demonstrated a block in this process in that BAX did not redistribute or homodimerize in response to a death signal. To test the functional consequence of BAX dimerization, we expressed a chimeric FKBP-BAX molecule. Enforced dimerization of FKBP-BAX by the bivalent ligand FK1012 resulted in its translocation to mitochondria and induced apoptosis. Caspases were activated yet caspase inhibitors did not block death; cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Moreover, enforced dimerization of BAX overrode the protection by BCL-XL and IL-3 to kill cells. These data support a model in which a death signal results in the activation of BAX. This conformational change in BAX manifests in its translocation, mitochondrial membrane insertion and homodimerization, and a program of mitochondrial dysfunction that results in cell death.
View details for Web of Science ID 000074980000009
View details for PubMedID 9670005
In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G1 and becomes hyper-phosphorylated in late G1. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15(INK4b) by transforming growth factor beta treatment of keratinocytes results in G1 arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15(INK4b)-independent transforming growth factor beta-mediated G1 arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16(INK4a) protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G1 allowing continued E2F binding.
View details for Web of Science ID A1997XY99800040
View details for PubMedID 9380698