Michael McMillan

Profibrotic Role of miR-154 in Pulmonary Fibrosis

In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly up-regulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-β1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-β was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/β-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 microRNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.

Investigating Wnt signaling: a chemogenomic safari.

Chemical genomics is a powerful method to complement more traditional genetic techniques (i.e. knockout mice, siRNA) for the dissection of complex signaling networks. Wnt signaling in mammals is a complex and crucial regulator of diverse functions. The Wnt-beta-catenin pathway initiates a signaling cascade that is crucial in both normal development and the initiation and progression of cancer. A key step in Wnt activation of target genes is the nuclear translocation of beta-catenin and the formation of a complex between beta-catenin and members of the T-cell factor (TCF) family of transcription factors. Using a forward chemical genomics strategy, we identified ICG-001, a selective inhibitor of a subset of Wnt-beta-catenin-driven gene expression. This chemogenomic tool enables us to dissect this complex signaling network and to better understand the role of Wnt signaling in both normal and pathophysiological settings.

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Wnt Signaling Orchestration with a Small Molecule DYRK Inhibitor Provides Long-Term Xeno-Free Human Pluripotent Cell Expansion

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno‐free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor‐β (TGFβ) signaling pathways, and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β‐catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor‐independent culture; however, the role of Wnt/β‐catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β‐catenin signaling in mouse embryonic stem cells, we identified a compound, ID‐8, that could support Wnt‐induced human embryonic stem cell proliferation and survival without differentiation. Dual‐specificity tyrosine phosphorylation‐regulated kinase (DYRK) is the target of the small molecule ID‐8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID‐8, we have developed a novel and simple chemically defined xeno‐free culture system that allows for long‐term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt‐dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.

Small Molecules and Stem Cells. Potency and Lineage Commitment: The New Quest for the Fountain of Youth

Regenerative Medicine and the Promise of Stem Cells The ability to control the regeneration of tissues or organs that do not normally regenerate in humans would have an enormous impact on medical practice as well as on the general quality of human life. The goal of regenerative medicine is to repair or replace damaged or diseased adult tissues or organs. Three general strategies are being investigated for regenerative therapies: 1) cell based; 2) engineered bio-scaffolds seeded with selected cells prior to engraftment; and 3) boosting endogenous repair mechanisms. Small molecule agents can potentially play critical roles as part of all three approaches. The cell-based approach has grown largely from the successful use of hematopoietic stem cell (HSC) transplants (bone marrow transplants) for more than 50 years now, primarily to treat blood disorders (for review1). Recently, the potential to significantly expand the scope of this approach has gained momentum from the ability to isolate adult multipotent stem cells from virtually all organs as well as from newly discovered capabilities to direct the differentiation of embryonic stem cells (ES) to multiple lineages. The recent advent to derive patient specific induced pluripotent cells (iPS) has further enhanced the potential of this approach by removing both ethical barriers and barriers to immuno-histocompatibility. The third approach has to this point relied heavily on the hope of recapitulating in mammals/humans some of the extremely impressive regenerative capacity of lower species (e.g. reptiles, amphibians, fish, and birds). However, the potential success of this approach is evidenced by the use of agents such as erythropoietin (EPO) and filgastrim (G-CSF) to boost hematopoietic recovery. These represent important clinically validated applications of this type of approach. The potential to expand this strategy and develop pharmacologic therapies to boost endogenous repair mechanisms for a wide array of tissues by overcoming the intrinsic barriers to regeneration in mammals remains an enormously exciting possibility. In this review, we will discuss the significant potential role that small molecules have to play in gaining a better understanding of stem cell biology, enabling regenerative therapies and treating stem cell based diseases.

Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors.

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel beta-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

Specific Direct Small Molecule p300/β-Catenin Antagonists Maintain Stem Cell Potency.

Despite their high degree of identity and even higher homology, the two Kat3 transcriptional coactivators, CBP and p300, have distinct functions, particularly within the Wnt/β-catenin signaling cascade. ICG-001, by directly binding to CBP but not p300, inhibits CBP/β-catenin transcription and has served as an invaluable chemical genomic tool to dissect the Wnt signaling cascade and the divergent roles of these two coactivators. However, to date no direct antagonist of the p300/β-catenin interaction has been reported. We now report the identification and validation of the first highly specific, direct p300/β-catenin antagonists, YH249/250 and their ability to maintain pluripotency in ESC.

Abstract B218: CBP/catenin antagonism inhibits self-renewal of acute lymphoblastic leukemia.

Background: Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), drug resistance in ALL remains a major problem warranting new treatment strategies. Recent studies have demonstrated that survivin, a member of the inhibitor of apoptosis (IAP) family proteins, is upregulated in ALL of relapsed patients but not in drug-sensitive ALL. The expression of survivin depends on the formation of a complex between catenin (beta or gamma) and its co-activator CBP. T-cell factor (TCF)/catenin/CBP mediated transcription is involved in self-renewal of progenitor cells. However, a switch to TCF/catenin/p300 mediated transcription, which can be induced pharmacologically with ICG-001, a novel specific inhibitor of binding to the N-terminus of CBP, is described as critical to inhibit CBP-mediated self-renewal. We hypothesized that selective suppression of CBP/catenin signaling using ICG-001 offers a promising therapeutic principle to eradicate drug resistant ALL and showed previously that ICG-001 can sensitize patient-derived (primary) pre-B ALL cells to chemotherapy in vitro and in vivo. However, the underlying mechanism has not been described. Results: For this purpose, we initially determined expression of nuclear and cytosolic β- and γ-catenin expression in 13 primary ALL cases encompassing various cytogenetic aberrations by Western blot. Primary pre-B ALL cells were treated with and without ICG-001 and co-cultured with a murine stromal layer (OP9 cells) for 48 hours. Co-Immunoprecipitation showed that the binding of β- and γ-catenin with CBP, but not with p300, was inhibited by ICG-001 indicating that ICG-001 binds CBP and selectively disrupts β- and γ-catenin/CBP interaction. To determine the effect of ICG-001 on self-renewal in ALL, we performed a colony forming (CFU) assay with two primary ALL cells (LAX7R, SFO2) treated with single agent ICG-001 (10 m and 25 m). LAX7R showed a significant reduction with ICG-001 treatment (10 m and 25 m) compared with media only group (331.3× 42.8 and 57×13.5 vs 698.3× 68.4; p Preclinical in vitro evaluation of five primary ALL cells treated continuously with ICG-001 (10μM- or, 25μM) or DMSO as vehicle control with and without chemotherapy consisting of Nilotinib for Philadelphia chromosome (Ph) positive ALL and Vincristine, Dexamethasone and L-Asparaginase for Ph negative ALL, that chemotherapy alone cannot eradicate primary ALL cells. However, in combination with ICG-001, chemotherapy led to the eradication of all five primary ALL cases, indicating that CBP/catenin antagonism can overcome drug resistance in ALL. Conclusion: Taken together, our data shows that targeting CBP/β- and γ-catenin by the small molecule inhibitor ICG-001 can abrogate primary ALL by inhibition of self-renewal of ALL cells. These findings provide the basis for a new treatment approach against drug resistant ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B218.