Amir Faisal

Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA

Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The conventional approach for studying individual isoforms in a clean background (i.e. without the influence of other isoforms) has been to express them in cells or whole organisms in which the target gene has been deleted; this is time-consuming. Recently an efficient post-transcriptional gene-silencing method has been reported that employs a small interfering double-stranded RNA (siRNA). On the basis of this method we report a rapid alternative approach for isoform-specific gene expression. We show how the adaptor protein ShcA can be suppressed and expressed in an isoform-specific manner in a human cell line. ShcA exists in three isoforms, namely p66, p52 and p46, which differ only in their N-terminal regions and are derived from two different transcripts, namely p66 and p52/p46 mRNAs. An siRNA with a sequence shared by the two transcripts suppressed all of them. However, another siRNA whose sequence was present only in p66 mRNA suppressed only the p66 isoform, suggesting that the siRNA signal did not propagate to other regions of the target mRNA. The expression of individual isoforms was achieved by first down-regulating all isoforms by the common siRNA and then transfecting with an expression vector for each isoform that harboured silent mutations at the site corresponding to the siRNA. This allowed functional analysis of individual ShcA isoforms and may be more generally applicable for studying genes encoding multiple proteins.

The regulated assembly of a PKC|[epsiv]| complex controls the completion of cytokinesis

The cell cycle is exquisitely controlled by multiple sequential regulatory inputs to ensure fidelity. Here we demonstrate that the final step in division, the physical separation of daughter cells, is controlled by a member of the PKC gene superfamily. Specifically, we have identified three phosphorylation sites within PKCɛ that control its association with 14-3-3. These phosphorylations are executed by p38 MAP kinase (Ser 350), GSK3 (Ser 346) and PKC itself (Ser 368). Integration of these signals is essential during mitosis because mutations that prevent phosphorylation of PKCɛ and/or PKCɛ binding to 14-3-3 also cause defects in the completion of cytokinesis. Using chemical genetic and dominant-negative approaches it is shown that selective inhibition of PKCɛ halts cells at the final stages of separation. This arrest is associated with persistent RhoA activation at the midbody and a delay in actomyosin ring dissociation. This study therefore identifies a new regulatory mechanism that controls exit from cytokinesis, which has implications for carcinogenesis.

Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: A model for emerging clinical resistance patterns

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

p66shc Inhibits Pro-survival Epidermal Growth Factor Receptor/ERK Signaling during Severe Oxidative Stress in Mouse Renal Proximal Tubule Cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66shc isoform can inhibit this p46/52shc function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser36 phosphorylation of p66shc. Isoform-specific knockdown of p66shc or mutation of Ser36 to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66shc to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser36 phosphorylation of p66shc and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser36 phosphorylation of p66shc or knocking down p66shc expression in vivo.

Imidazo[4,5-b]pyridine Derivatives As Inhibitors of Aurora Kinases: Lead Optimization Studies toward the Identification of an Orally Bioavailable Preclinical Development Candidate

Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss.

p66SHC-mediated mitochondrial dysfunction in renal proximal tubule cells during oxidative injury

Mitochondrial dysfunction is involved in pathopysiology of ischemia-reperfusion-induced acute kidney injury (AKI). The p66shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction, which might play a role in AKI-induced renal tubular injury. Oxidative stress-mediated Serine36 phosphorylation of p66shc facilitates its transportation to the mitochondria where it oxidizes cytochrome c and generates excessive amount of reactive oxygen species (ROS). The consequence is mitochondrial depolarization and injury. Earlier we determined that p66shc plays an essential role in injury of cultured mouse renal proximal tubule cells during oxidative stress. Here, we studied the role of p66shc in ROS generation and consequent mitochondrial dysfunction during oxidative injury in renal proximal tubule cells. We employed p66shc knockdown renal proximal tubule cells and cells that overexpress wild-type, Serine phosphorylation (S36A), or cytochrome c-binding (W134F) mutants of p66shc. Inhibition of the mitochondrial electron transport chain or the mitochondrial permeability transition revealed that hydrogen peroxide-induced injury is mitochondrial ROS and consequent mitochondrial depolarization dependent. We also found that through Ser36 phosphorylation and mitochondria/cytochrome c binding, p66shc mediates those effects. We propose a similar mechanism in vivo as we demonstrated mitochondrial binding of p66shc as well as its association with cytochrome c in the postischemic kidneys of mice. Thus, manipulating p66shc might offer a new therapeutic modality to ameliorate renal ischemic injury.

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

The Scaffold MyD88 Acts to Couple Protein Kinase Cϵ to Toll-like Receptors

Mice lacking protein kinase Cϵ (PKCϵ) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCϵ coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCϵ involvement in TLR signaling and found that PKCϵ is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCϵ binding to 14-3-3β. LPS-induced PKCϵ phosphorylation, 14-3-3β binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCϵ phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCϵ association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCϵ. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCϵ was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCϵ at these two sites is required for TLR4- and TLR2-induced NFκB reporter activation and IκB degradation in reconstituted PKCϵ–/– cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCϵ recruitment, phosphorylation, and downstream signaling.

The Aurora Kinase Inhibitor CCT137690 Downregulates MYCN and Sensitizes MYCN-Amplified Neuroblastoma In Vivo

Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora A and B kinase overexpression has also been associated with various human cancers, and as such, they have been extensively studied as novel antimitotic drug targets. Here, we characterize the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC50 values in both biochemical and cellular assays and exhibits antiproliferative activity against a wide range of human solid tumor cell lines. CCT137690 efficiently inhibits histone H3 and transforming acidic coiled-coil 3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumor cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy, and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 downregulation, and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumor growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN-amplified neuroblastoma. Mol Cancer Ther; 10(11); 2115–23. ©2011 AACR.

Aurora Isoform Selectivity: Design and Synthesis of Imidazo[4,5-B]Pyridine Derivatives as Highly Selective Inhibitors of Aurora-A Kinase in Cells.

Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand–Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells.