Distinct mobilization of leukocytes and hematopoietic stem cells by CXCR4 peptide antagonist LY2510924 and monoclonal antibody LY2624587.

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy.

Oncogenic BRAF Deletions That Function as Homodimers and Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120.

UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

A Novel Eg5 Inhibitor (LY2523355) Causes Mitotic Arrest and Apoptosis in Cancer Cells and Shows Potent Antitumor Activity in Xenograft Tumor Models.

Intervention of cancer cell mitosis by antitubulin drugs is among the most effective cancer chemotherapies. However, antitubulin drugs have dose-limiting side effects due to important functions of microtubules in resting normal cells and are often rendered ineffective by rapid emergence of resistance. Antimitotic agents with different mechanisms of action and improved safety profiles are needed as new treatment options. Mitosis-specific kinesin Eg5 represents an attractive anticancer target for discovering such new antimitotic agents, because Eg5 is essential only in mitotic progression and has no roles in resting, nondividing cells. Here, we show that a novel selective Eg5 inhibitor, LY2523355, has broad target-mediated anticancer activity in vitro and in vivo. LY2523355 arrests cancer cells at mitosis and causes rapid cell death that requires sustained spindle-assembly checkpoint (SAC) activation with a required threshold concentration. In vivo efficacy of LY2523355 is highly dose/schedule-dependent, achieving complete remission in a number of xenograft tumor models, including patient-derived xenograft (PDX) tumor models. We further establish that histone-H3 phosphorylation of tumor and proliferating skin cells is a promising pharmacodynamic biomarker for in vivo anticancer activity of LY2523355.

A novel CDK9 inhibitor shows potent antitumor efficacy in preclinical hematologic tumor models.

DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.

Multiple dopamine receptors mediate the discriminative stimulus effects of dihydrexidine in rats.

The dopamine D(1) receptor agonist dihydrexidine (DHX) [(±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a] phenanthridine hydrochloride] has shown efficacy in animal models of Parkinson's disease and improved cerebral blood flow and working memory of schizophrenic patients. Although the discriminative stimulus effects of DHX, an in-vivo predictor of human subjective effect profile, have only been characterized with respect to activity at D(1) receptors, DHX also has significant affinity for D(2) receptors. This study was designed to characterize the role of D(1) and D(2)/D(3) receptors in mediating the discriminative stimulus effects of DHX. Rats were trained to discriminate DHX [3 mg/kg, intraperitoneally (i.p.)] from the vehicle. The selective dopamine D(1) receptor partial agonist SKF 38393 was fully substituted for DHX. The D(1) receptor antagonist SCH 23390 (0.1 mg/kg, s.c.) and the D(3)-selective antagonist U99194 (10 mg/kg, i.p.) significantly attenuated the discriminative stimulus effects of the training dose of DHX by 80 and 60%, respectively, suggesting that both D(1) and D(3) receptors mediate the discriminative stimulus effects of DHX. In contrast, raclopride (1 mg/kg, i.p.) did not significantly alter the discriminative stimulus effects of DHX, indicating a lack of D(2)-mediated effects. The D(2)/D(3) receptor preferring agonists, quinpirole and (+)-PD 128907 were fully substituted, whereas (+)-7-OH-DPAT was partially substituted for DHX. The DHX bound to D(2) receptors with a Ki of 4.3+0.7 nmol/l was compared with 33.7+4.6 nmol/l at D(3) receptors. Determinations of activity at second messenger systems revealed that DHX functioned as a full agonist at D(3) receptors and a partial agonist at D(2) receptors in vitro. These activities at D(2)/D(3) receptors have shown effects in some preclinical models and clinical disease states. Therefore, the prominent in-vivo agonist activity of DHX at both D(1) receptors and D(2)/D(3) receptors should be considered while making predictions of effects in humans.

Cdk1 activity is required for mitotic activation of aurora A during G2/M transition of human cells.

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

Pharmacologic characterization of the cloned human trace amine-associated receptor1 (TAAR1) and evidence for species differences with the rat TAAR1.

The hemagglutinin-tagged human trace amine-associated receptor1 (TAAR1) was stably coexpressed with rat Galpha(s) in the AV12-664 cell line, and receptor activation was measured as the stimulation of cAMP formation. After blockade of endogenously expressed alpha2- and beta-adrenoceptors with 2-[2-(2-methoxy-1,4-benzodioxanyl)]-imidazoline hydrochloride (2-methoxyidazoxan, RX821002) and alprenolol, respectively, the resulting pharmacology was consistent with that of a unique receptor subtype. beta-Phenylethylamine (beta-PEA), the putative endogenous ligand, gave an EC50 of 106 +/- 5 nM in the assay. For a series of beta-PEA analogs used to explore the pharmacophore, small substituents at ring positions 3 and/or 4 generally resulted in compounds having lower potency than beta-PEA, although several were as potent as beta-PEA. However, small substituents at ring position 2 resulted in a number of compounds having potencies as good as or better than beta-PEA. A number of nonselective antagonists known to share affinity for multiple monoaminergic receptors were evaluated for their ability to inhibit beta-PEA stimulation of the human TAAR1. None had an IC50 <10 microM. For comparison, the rat TAAR1 receptor was expressed in the AV12-664 cell line. A number of agonist compounds had significantly different relative potencies between the rat and human TAAR1, demonstrating a significant species difference between the rat and human TAAR1. The TAAR1 receptor exhibits a pharmacologic profile uniquely different from those of classic monoaminergic receptors, consistent with the structural information that places them in a distinct family of receptors. This unique pharmacologic profile suggests the potential for development of TAAR-selective agonists and antagonists to study their physiologic roles.

Molecular cloning and pharmacological characterization of the guinea pig 5-HT1E receptor.

The human 5-HT(1E) receptor gene was cloned more than a decade ago. Little is known about its function, and there have been no reports of its existence in the genome of small laboratory animals. In this study, attempts to clone the 5-HT(1E) gene from the rat and mouse were unsuccessful. In fact, a search of the mouse genome database revealed that the 5-HT(1E) receptor gene is missing from the mouse genome. However, the 5-HT(1E) gene was cloned from guinea pig genomic DNA and was characterized. The guinea pig 5-HT(1E) receptor gene encodes a protein of 365 amino acids. It shares 88% (nucleic acid) and 95% (amino acid) homology with the human receptor. The guinea pig 5-HT(1E) receptor showed similar pharmacology to the human 5-HT(1E) receptor in radioligand binding assays. Serotonin (5-hydroxytryptamine, 5-HT) dose-dependently stimulated [35S]GTPgammaS binding to the guinea pig 5-HT(1E) receptor with an EC(50) of 13.6+/-1.92 nM, similar to that of the human 5-HT(1E) receptor (13.7+/-1.78 nM). Activation of the guinea pig 5-HT(1E) receptor was also achieved by ergonovine, alpha-methyl-5-HT, 1-naphthylpiperazine, methysergide, tryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Methiothepin exhibited antagonist activity. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 5-HT(1E) mRNA was present in the guinea pig brain with the greatest abundance in the hippocampus, followed by the olfactory bulb. Lower levels were detected in the cortex, thalamus, pons, hypothalamus, midbrain, striatum, and cerebellum. Our current study marks the first identification of the 5-HT(1E) receptor gene in a commonly used laboratory animal species. This finding should allow the elucidation of the receptor's role(s) in the complex coordination of central serotonergic effects.

Specificity of interleukin-2 receptor gamma chain superfamily cytokines is mediated by insulin receptor substrate-dependent pathway.

Interleukins 9 (IL-9) and 4 are cytokines within the IL-2 receptor gamma chain (IL-2R gamma) superfamily that possess similar and unique biological functions. The signaling mechanisms, which may determine cytokine specificity and redundancy, are not well understood. IRS proteins are tyrosine-phosphorylated following IL-9 and IL-4 stimulation, a process in part mediated by JAK tyrosine kinases (Yin, T. G., Keller, S. R., Quelle, F. W., Witthuhn, B. A., Tsang, M. L., Lienhard, G. E., Ihle, J. N., and Yang, Y. C. (1995) J. Biol. Chem. 270, 20497--20502). In the present study, we used 32D cells stably transfected with insulin receptor (32D(IR)), which do not express any IRS proteins, as a model system to study the requirement of different structural domains of IRS proteins in IL-9- and IL-4-mediated functions. Overexpression of IRS-1 and IRS-2, but not IRS-4, induced proliferation of 32D(IR) cells in response to IL-9. The pleckstrin homology (PH) domain of IRS proteins is required for IRS-mediated proliferation stimulated by IL-9. The phosphotyrosine binding and Shc and IRS-1 NPXY binding domains are interchangeable for IRS to transduce the proliferative effect of IL-4. Therefore, the PH domain plays different roles in coupling IRS proteins to activated IL-9 and IL-4 receptors. The role of IRS proteins in determining cytokine specificity was corroborated by their ability to interact with different downstream signaling molecules. Although phosphatidylinositol 3' -kinase (PI3K) and Grb-2 interact with tyrosine-phosphorylated IRS proteins, Shp-2 only binds to IRS proteins following IL-4, but not IL-9, stimulation. Although PI3K activity is necessary for the IRS-1/2-mediated proliferative effect of IL-9 and IL-4, Akt activation is only required for cell proliferation induced by IL-4, but not IL-9. These data suggest that IRS-dependent signaling pathways work by recruiting different signaling molecules to determine specificity of IL-2R gamma superfamily cytokines.