Molecule Protein Kinase Inhibitors Research Articles

Small Molecule Inhibitor of Protein Kinase C DeltaI (PKCδI) Decreases Inflammatory Pathways and Gene Expression and Improves Metabolic Function in Diet-Induced Obese Mouse Model.

Obesity promotes metabolic diseases such as type 2 diabetes and cardiovascular disease. PKCδI is a serine/threonine kinase which regulates cell growth, differentiation, and survival. Caspase-3 cleavage of PKCδI releases the C-terminal catalytic fragment (PKCδI_C), which promotes inflammation and apoptosis. We previously demonstrated an increase in PKCδI_C in human obese adipose tissue (AT) and adipocytes. Subsequently, we designed a small molecule drug called NP627 and demonstrated that NP627 specifically inhibited the release of PKCδI_C in vitro. Here, we evaluate the in vivo safety and efficacy of NP627 in a diet-induced obese (DIO) mouse model. The results demonstrate that NP627 treatment in DIO mice increased glucose uptake and inhibited the cleavage of PKCδI_C in the AT as well as in the kidney, spleen, and liver. Next, RNAseq analysis was performed on the AT from the NP627-treated DIO mice. The results show increases in ADIPOQ and CIDEC, upregulation of AMPK, PI3K-AKT, and insulin signaling pathways, while inflammatory pathways were decreased post-NP627 administration. Further, levels of lncRNAs associated with metabolic pathways were affected by NP627 treatment. In conclusion, the study demonstrates that NP627, a small-molecule inhibitor of PKCδI activity, is not toxic and that it improves the metabolic function of DIO mice in vivo.

Asymmetric Synthesis of CIDD-0072424 via an Enantioselective Nitro-Mannich Reaction: A Central Nervous System Penetrant, Selective Small Molecule Inhibitor of Protein Kinase C Epsilon.

CIDD-0072424 is a novel small molecule developed in silico with remarkable activity for the inhibition of protein kinase C (PKC)-epsilon to treat alcohol use disorder. We developed a concise synthesis of (S)-2 that is highly enantioselective, scalable, and amenable for 3-point structure-activity relationship (SAR) studies for compound optimization. The highly enantioselective nitro-Mannich reaction was achieved through a dual-reagent catalysis system. The overall utility and the efficiency of the enantioselective route provided a scalable synthesis of both PKCε inhibitors 1 and 2.

Natural Products in Precision Oncology: Plant-Based Small Molecule Inhibitors of Protein Kinases for Cancer Chemoprevention.

Striking progress is being made in cancer treatment by using small molecule inhibitors of specific protein kinases that are products of genes recognized as drivers for a specific type of cancer. However, the cost of newly developed drugs is high, and these pharmaceuticals are neither affordable nor accessible in most parts of the world. Accordingly, this narrative review aims to probe how these recent successes in cancer treatment can be reverse-engineered into affordable and accessible approaches for the global community. This challenge is addressed through the lens of cancer chemoprevention, defined as using pharmacological agents of natural or synthetic origin to impede, arrest, or reverse carcinogenesis at any stage in the disease process. In this regard, prevention refers to reducing cancer-related deaths. Recognizing the clinical successes and limitations of protein kinase inhibitor treatment strategies, the disciplines of pharmacognosy and chemotaxonomy are juxtaposed with current efforts to exploit the cancer kinome to describe a conceptual framework for developing a natural product-based approach for precision oncology.

Small Molecule Inhibitors of Protein Kinase D: Early Development, Current Approaches, and Future Directions.

Now entering its fourth decade, research on the biological function, small molecule inhibition, and disease relevance of the three known isoforms of protein kinase D, PKD1, PKD2, and PKD3, has entered a mature development stage. This mini-perspective focuses on the medicinal chemistry that provided a structurally diverse set of mainly active site inhibitors, which, for a brief time period, moved through preclinical development stages but have yet to be tested in clinical trials. In particular, between 2006 and 2012, a rapid expansion of synthetic efforts led to several moderately to highly PKD-selective chemotypes but did not yet achieve PKD subtype selectivity or resolve general toxicity and pharmacokinetic challenges. In addition to cancer, other unresolved medical needs in cardiovascular, inflammatory, and metabolic diseases would, however, benefit from a renewed focus on potent and selective PKD modulators.

Light-Control over Casein Kinase 1δ Activity with Photopharmacology: A Clear Case for Arylazopyrazole-Based Inhibitors.

Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.

Decorin-An Antagonist of TGF-β in Astrocytes of the Optic Nerve.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis.

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

Cardiovascular side effects of small molecule therapies for cancer.

Targeted therapies such as monoclonal antibodies and small molecule inhibitors of protein kinases, have improved the prognosis for patients with a wide range of cancers. Several of these classes of therapies are associated with cardiovascular and metabolic sequelae. These include heart failure or an asymptomatic decline in ejection fraction, peripheral and cardiac ischemic events, pulmonary hypertension, arrhythmias and QT-prolongation. This review will focus on the following targeted therapies: BCR-Abl tyrosine kinase inhibitors (TKI), vascular endothelial growth factor (VEGF) signaling pathway (VSP) inhibitors as well as mitogen activated protein kinase (MEK) inhibitors and mammalian target of rapamycin complex (mTOR) inhibitors. The HER2/ErbB2 inhibitors trastuzumab and pertuzumab are important targeted therapies used in the treatment of breast cancer that have been reviewed in detail elsewhere and will not be discussed in this review.1 Table 1 summarizes the cardiovascular side effects of small molecule therapies for cancer.2 View this table: Table 1 Cardiovascular Side effects of Small Molecule Therapies for Cancer BCR-Abl tyrosine kinase inhibitors are an important advance in the treatment of chronic myelogenous leukemia (CML) and the cardiovascular side effects have been extensively reviewed by Moslehi et al.3 Imatinib, a first generation TKI was followed by the second generation TKIs nilotinib, dasatinib and bosutinib and the third-generation agent ponatinib. Imatinib has a low incidence of cardiomyopathy and heart failure, with sequential imaging studies demonstrating rates of left ventricular dysfunction comparable to expected population incidence.4 The heart failure and cardiomyopathy rates for dasatinib were similar …

Sa1480 Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-κB Activation in In Vitro and In Vivo Experimental Acute Pancreatitis

Sa1480 Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-κB Activation in In Vitro and In Vivo Experimental Acute Pancreatitis

Quantitative proteomics of kinase inhibitor targets and mechanisms.

Small molecule inhibitors of protein kinases are key tools for signal transduction research and represent a major class of targeted drugs. Recent developments in quantitative proteomics enable an unbiased view on kinase inhibitor selectivity and modes of action in the biological context. While chemical proteomics techniques utilizing quantitative mass spectrometry interrogate both target specificity and affinity in cellular extracts, proteome-wide phosphorylation analyses upon kinase inhibitor treatment identify signal transduction pathway and network regulation in an unbiased manner. Thus, critical information is provided to promote new insights into mechanisms of kinase signaling and their relevance for kinase inhibitor drug discovery.

The Reciprocal Interaction of Small Molecule Protein Kinase Inhibitors and ATP-Binding Cassette Transporters in Targeted Cancer Therapy

Protein kinaseshave become the second most important group of drug targets, after G-protein-coupled receptors. Currently,15 small molecule protein kinase inhibitors (PKIs) have received food and drug administrator (FDA) approval to be used as cancer treatments. However, in the course of clinical use of these small molecule PKIs, drug resistance has become a recurring problem. Their therapeutic potential depends on access to their intracellular targets, which significantly affected by certain membrane ATP-binding cassette(ABC) transporters. ABC transporters were major causesof clinical multiple drug resistance (MDR) and might be resulting in the development of resistance to PKIs in cancer patients. Some PKIs could modulate the activity of ABC transporters and affect the metabolism of themselves and other chemically unrelated drugs. Moreover, it has been recently reported that some PKIs could regulate the expression of ABC transporters in tumor cells, thereby affect their intracellular accumulation and antitumor efficacy. In this review,the reciprocal interaction of clinically important PKIs with the MDR-related ABC transporters, in particular ABCB1 and ABCG2,wassummarized. Keyword:Protein Kinase Inhibitors, ABC Transporters, P-gp/ABCB1, BCRP/ABCG2, Targeted Cancer Therapy.

A-Kinase-Anchoring Protein–Lbc Connects Stress Signaling to Cardiac Hypertrophy

Heart failure often occurs as a consequence of persistent trauma to the myocardium. Cardiovascular pathologies, including hypertension, valvular disease, atherosclerosis, and ischemia, can all lead to pressure overload and myocardial dysfunction. In the face of such stressors, the heart attempts to maintain normal contractile function by initiating a complex remodeling process involving the reexpression of developmental genes. This process leads to an increase in cardiac muscle mass commonly referred to as pathological cardiac hypertrophy. If trauma is persistent or severe, such compensatory mechanisms are overwhelmed or become maladaptive and heart failure ensues. Stress signaling pathways play an important role in cellular responses to cardiotoxic insults such as mechanical shear and an overabundance of proinflammatory molecules. Moreover, recent development of effective drug therapies targeting these pathways has renewed interest in the role of stress signaling in ventricular cardiomyocyte hypertrophy (1, 2). A variety of well-known signaling cascades underlie the onset of cardiomyocyte hypertrophy. Although the precise molecular details of these pathways are not clear, several groups have uncovered valuable clues that point to the complexity of the signaling events that lead to these phenotypes (3). Adrenergic agonists, acting through 1-adrenergic receptors ( 1-AR) and G 12, activate the small G protein RhoA, which then engages both the Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) kinase cascades (4–6). These studies point toward stress signaling as a major contributor to the hypertrophic response. In this issue, del Vescovo et al. describe an intriguing new connection between adrenergic, small GTPase, and cytokine signaling that regulates stress effects on cardiac remodeling (7). del Vescovo and colleagues have identified a robust protein-protein interaction between A-kinase-anchoring protein (AKAP)–Lbc and I B kinase (IKK ), a crucial regulator of NFB signaling. Interestingly, AKAP-Lbc is an AKAP that also possesses Rho guanine nucleotide exchange factor (GEF) activity and acts as a scaffold for multiple kinases involved in cardiomyocyte function (5, 8, 9). In this context, AKAP-Lbc promotes fetal gene reprogramming through a protein kinase D (PKD)-histone deacetylase 5 (HDAC5) pathway (10) and functions downstream of 1-adrenergic receptors to activate G 12-mediated RhoA signaling (5). Through a combination of mass spectrometry and standard biochemical analyses, del Vescovo and colleagues showed that IKK binds to AKAP-Lbc. This stress-activated kinase phosphorylates and targets I B for proteasomal degradation, releasing the transcription factor NFB from inhibition and allowing it to enter the nucleus (11). Once in the nucleus, NFB initiates a predetermined program of gene expression to combat cardiac stresses. More-detailed biochemical mapping experiments identified a short helical region at the end of the AKAP-Lbc pleckstrin homology (PH) domain that was responsible for interaction with IKK . Furthermore, a point mutation in AKAP-Lbc, W2328L, dramatically reduces IKK binding (7). Next, del Vescovo et al. showed that short hairpin RNA (shRNA)-mediated silencing of AKAPLbc impairs activation of an NFB reporter gene. Silencing AKAP-Lbc expression also reduced phenylephrine (PE)-induced IKK kinase activity. Thus, the anchoring of IKK by AKAP-Lbc permits transmission of adrenergic signals to NFB. A previous report had implicated the RhoA effector Rho kinase as an activator of NFB (12). Consequently, del Vescovo et al. asked whether an AKAP-Lbc-associated RhoA pathway relayed signals to NFB. Using an AKAP-Lbc mutant with constitutive Rho GEF activity, they demonstrated that application of the Rho kinase inhibitor Y27632 impairs NFB transcriptional activity. This inhibitor also blocks AKAP-Lbc-mediated activation of IKK , as assessed by in vitro kinase assays. Moreover, these effects appear to be specific for the Rho pathway, as small molecule inhibitors of protein kinase C (PKC ), p38 , and MEK1, which are other kinases that associate with AKAP-Lbc, had no effects on the NFB transcriptional reporter (4, 7, 13). Finally, del Vescovo et al. demonstrated a requirement for an AKAP-Lbc/IKK subcomplex to initiate NFB transcription, as RNA interference (RNAi) rescue experiments were ineffective upon reexpression of the AKAP-Lbc W2328L mutant, which no longer anchors IKK (7). Thus, adrenergic and stress signaling pathways seem to converge at the level of the AKAP-Lbc signaling complex. While the work of del Vescovo and colleagues provides clear evidence of a link between the adrenergic and stress signaling pathways in myocytes, several key questions still remain. For example, how does adrenergic signaling to NFB result in cardiomyocyte hypertrophy? One clue was provided by recent work demonstrating that 1-adrenergic signals promote expression of the cytokine interleukin-6 (IL-6) in an NFB-dependent manner (14). Importantly, del Vescovo et al. were also able to show that inhibition of IL-6 signaling impairs 1-AR-mediated induction of fetal genes, as indicated by atrial natriuretic factor (ANF) and -myosin heavy chain ( -MHC) gene transcription. Taken together, these data suggest that IL-6 is produced and secreted downstream of AKAP-Lbc/RhoA/NFB, where it acts in a paracrine/autocrine manner to promote transcription of fetal genes characteristic of the hypertrophic response (Fig. 1). It will be in-

Novel Protein Kinase Signaling Systems Regulating Lifespan Identified by Small Molecule Library Screening Using Drosophila

Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells

Small molecule inhibitors of protein kinases have emerged as a major class of therapeutic agents for the treatment of hematological malignancies. Both in vitro studies and patient case reports suggest therapeutic potential of the clinical kinase inhibitors erlotinib and gefitinib in acute myeloid leukemia (AML). The drugs' cellular modes of action in AML warrant further investigation as their primary therapeutic target, the epidermal growth factor receptor, is not expressed. We therefore performed SILAC-based quantitative mass spectrometry analyses to a depth of 10,975 distinct phosphorylation sites to characterize the phosphoproteome of KG1 AML cells and its regulation upon erlotinib and gefitinib treatment. Less than 50 site-specific phosphorylations changed significantly, indicating rather specific interference with AML cell signaling. Many drug-induced changes occurred within a network of tyrosine phosphorylated proteins that included Src family kinases (SFKs) and the tyrosine kinases Btk and Syk. We further performed quantitative chemical proteomics in KG1 cell extracts and identified SFKs and Btk as direct cellular targets of both erlotinib and gefitinib. Taken together, our data suggest that cellular perturbation of SFKs and/or Btk translates into rather specific signal transduction inhibition, which in turn contributes to the antileukemic activity of erlotinib and gefitinib in AML.

414 A Novel Small Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth Both In Vitro and In Vivo

414 A Novel Small Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth Both In Vitro and In Vivo

Abstract B261: A novel small molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vivo

Abstract Background: Protein kinase D (PKD) is a novel family of serine-threonine kinase with diverse biological functions including cell proliferation and growth. Pancreatic Cancer (PaCa) is a devastating disease with few therapeutic options. We showed earlier that PKD signaling pathways promote mitogenesis in multiple PaCa cell lines. However, nothing is known about targeting biological functions of PKD in PaCa. Our PKD inhibitor discovery program yielded CRT0066101 that specifically blocks PKD activation. Aim: The aim of our study was to determine the effects of CRT0066101 in PaCa, both in vitro and in vivo. Methods and Results: Our immunohistochemical analysis showed that activated PKD (pS916PKD1) is significantly upregulated in PaCa as compared to normal ducts (91% vs 22%; p<0.001). Using Panc-1 as a model system, we demonstrated that CRT0066101 blocked proliferation and BrdU incorporation with an IC50 of 1 µM. We showed that CRT0066101 given orally (80 mg/kg/day) for 4 weeks significantly abrogated growth in a subcutaneous Panc-1 xenograft model (n=8; p<0.01). The expression of activated PKD (pS916PKD1) in the treated tumor explants was significantly inhibited (p<0.05) with peak concentration (12 M) of CRT0066101 achieved within 6 hours of oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/day) for 21 days in an orthotopic model potently blocked Panc-1 tumor growth (n=7; p<0.01). CRT0066101 significantly reduced Ki-67+ proliferation index (p< 0.01), increased apoptosis (measured by in situ TUNEL assay) of PaCa tumors (p<0.05) and potently abrogated expression of multiple proliferative and pro-survival proteins including Cyclin D1, survivin, Bcl-2, Bcl-xL, activated PKD1 (pS916PKD1), and activated PKD2 (pS876PKD2). Conclusion: Our results demonstrate for the first time that the PKD-specific small molecule inhibitor CRT0066101 blocks PaCa growth both in vitro and in vivo. Thus, PKD is a novel therapeutic target in PaCa. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B261.

CRLF2/JAK Signaling Confers Susceptibility to JAK Inhibitors and Small Molecule Inhibitors of Protein Kinase C.

Abstract 3767Poster Board III-703 IntroductionWe and others recently identified the type I-like cytokine receptor CRLF2 as an oncoprotein in adult and high-risk pediatric precursor B-cell acute lymphoblastic leukemia (B-ALL). Wild-type CRLF2 heterodimerizes with IL7R and signals in response to thymic stromal lymphopoietin (TSLP) through unclear mechanisms that include STAT5 activation. In cases of B-ALL that overexpress CRLF2, the CRLF2 locus is rearranged through either a translocation with IGH or an intrachromosomal deletion. CRLF2 rearrangements are confined to patients who lack translocations involving MLL, E2A, TEL, and BCR/ABL. A subset of cases with CRLF2 overexpression also harbors a CRLF2 Phe232Cys gain-of-function mutation, while a separate subset has mutations in Janus Kinases (JAK). Cells transformed by CRLF2 Phe232Cys or the combination of wild-type CRLF2 with JAK2 exon 16 mutants have constitutive activation of STAT5 and ERK, and upregulation of STAT target genes. However, only the cells that express JAK2 mutants have constitutive phosphorylation of JAK2, suggesting that CRLF2 Phe232Cys signals through an alternate kinase. We used transcriptional profiling to identify candidate pathways and then screened available kinase inhibitors for selective toxicity in cells transformed by CRLF2 and JAK2 mutant alleles. MethodsWe reviewed transcriptional profiles obtained from 22 adult patients with B-ALL enrolled on Gruppo Malattie Ematologiche dell'Adulto (GIMEMA) protocols LAL2000 and LAL2004. All samples were confirmed to have high (n=8) or low (n=14) CRLF2 expression by quantitative RT-PCR. Samples were assayed on the Affymetrix HG-U133A platform, which contains a single probe set (208303_s_at) that targets the CRLF2 transcript (both complete and partial coding sequence, as well as expression sequence tags). Supervised analysis was performed using a t-test: probe sets were 1.5, and expression≥0.005 and fold change ≤required to have a p-value >100 in at least one group. For the kinase inhibitor screen, BaF3 cells that stably express CRLF2, IL7R and/or JAK2 alleles were incubated with compound for 48 hours in the absence of IL3. Viability was measured by ATP content with luminescence using CellTiterGlo and read on an Envision plate reader. ResultsSupervised analysis defined a 105 gene (130 probe set) signature associated with CRLF2 overexpression. This signature included upregulation of CD10, CD99, the STAT5 induced regulator SOCS2 and Protein Kinase C (PKC) iota. Downregulated genes included class I and class II major histocompatibility antigens. Based on the involvement of JAKs in CRLF2 signaling and the overexpression of PKC iota, we carried out a kinase inhibitor screen. Compared with cells that express wild-type CRLF2/IL7R grown in the presence of TSLP, cells transformed by CRLF2 Phe232Cys or wild-type CRLF2/mutant JAK2 had 10-100-fold lower EC50 for small-molecule inhibitors of JAK signaling and agents with activity against PKC family kinases (Go6976, UCN-01, k252a). Assays to confirm these findings in primary B-ALL specimens are underway. ConclusionCRLF2/JAK signaling confers a unique pattern of kinase inhibitor sensitivity that can be elucidated through transcriptional profiling and targeted with available agents. Disclosures:No relevant conflicts of interest to declare.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274

Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC 50 values for autophosphorylation inhibition ranging between 0.15 and 1.5 μM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCγ and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.

Small Molecule Inhibitors of Protein Kinases in Cancer- How to Overcome Resistance

Small molecule protein kinase inhibitors show great promise as anti-cancer agents, however, de novo and acquired resistance present problems. These are reviewed and illustrated using the receptor tyrosine kinase, KIT, as an example. Emerging solutions are presented, such as targeting active kinase conformations.

Chemical genetic methods for studying protein kinases

Our laboratory focuses on the development of novel chemically based tools to decipher signal transduction pathways on a genome-wide scale. We have developed a method for producing small molecules that are specific for any protein kinase of interest in a signaling cascade by combining protein design with chemical synthesis. These highly specific inhibitors of individual kinases have revealed a number of new principles of signal transduction that have complemented genetic and biochemical studies of cell signaling. Examples where new pathways and new functions can be revealed by small molecule inhibitors of protein kinases will be highlighted. A second area of interest in our laboratory is the tracing of direct kinase substrates. We have designed and synthesized unnatural ATP analogs, which are substrates of our engineered kinases but are poorly accepted as substrates of wild-type kinases. This specific nucleotide substrate of any kinase of interest allows for the radiolabeling of the direct substrates of a wide variety of protein kinases including both serine/threonine and tyrosine kinases. New methods for the isolation and identification of low abundance substrates of kinases from cells will be discussed. Once a phosphoprotein substrate of a kinase is identified, the specific phosphorylation site is often difficult to identify using traditional tryptic peptide phosphorylation site mapping. Using a novel strategy based on the design of tailor made proteases which specifically cleave proteins after sites of phosphorylation, we have developed a rapid means to map protein phosphorylation patterns. Finally, a potential link between the unnatural ligands of engineered kinases and a set of plant hormones, the cytokinins, will be discussed in the context of a custom designed database created for the genome wide analysis of protein kinase catalytic domains.