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BackgroundP21-activated kinase 4 (PAK4) is a serine/threonine protein kinase that is mostly expressed in tumor and stroma cells. PAK4 activates tumor WNT/β-catenin pathway and regulates cellular morphology, motility, EMT, cell proliferation and survival. Recent studies also showed that PAK4 can actively exclude T cells from tumors, suggesting that therapeutic inhibition of PAK4 can increase T cell infiltration in tumor microenvironment and overcome resistance to checkpoint inhibitor immunotherapy.1MethodsWe generated PAK4 knockout (KO) clones in human and mouse tumor cells to validate its biology in vitro and in vivo. We also performed pharmacological evaluation of PAK4 inhibition using Pfizer compounds (referred to as 'PAK4i compounds' below) for their potential tumor-intrinsic and immune-regulatory roles.ResultsNanostring, qPCR and RNASeq analysis showed that PAK4 depletion led to increase of cytokine expression in tumor, including conventional dendritic cell (cDC)- recruiting chemokine CCL4, and type I IFN / ISG pathway genes that are associated with MHC upregulation such as CXCL10. In addition, PAK4 KO sensitizes B16F10 tumors to anti-PD-1 treatment and increases infiltration of cDC and T cells in the tumor microenvironment.We also showed that small molecule PAK4i compounds induced more potent cancer cell growth inhibition over treated normal PBMCs. PAK4i compounds also increased immune-activating and decreased immune exclusion genes in B16F10 cells and tumor explants in vitro. Although PAK4 target engagement is demonstrated by CETSA assay, the compound potency on modulating PAK4 downstream Wnt/ β-catenin pathway is low, suggesting that the aforementioned phenotypic changes induced by PAK4i compounds may be partially attributed to other off-target effects.ConclusionsCollectively, our data suggests that genetic depletion or pharmacological inhibition of PAK4 may induce immune-activating cytokine production in tumor cells, revert immune cell exclusion in tumor microenvironment, and synergize with checkpoint blockade therapies. However, further optimization on these PAK4i compounds is needed to improve its specificity on modulating PAK4 enzyme activities.ReferenceAbril-Rodriguez G, Torrejon DY, Liu W, Zaretsky JM, Nowicki TS, Tsoi J, Puig-Saus C, Baselga-Carretero I, Medina E, Quist MJ, Garcia AJ, Senapedis W, Baloglu E, Kalbasi A, Cheung-Lau G, Berent-Maoz B, Comin-Anduix B, Hu-Lieskovan S, CWang CY, Grasso CS & Ribas A. PAK4 inhibition improves PD-1 blockade immunotherapy. Nat Cancer 2020;1:46–58.Ethics ApprovalAll animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Pfizer. Approved protocol # LAJ-2019-01347

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.

How type I and II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell types remains unknown. Using homogeneous neuron cultures latently infected with herpes simplex virus 1, we show that extrinsic type I or II interferon acts directly on neurons to induce unique gene expression signatures and inhibit the reactivation-specific burst of viral genome-wide transcription called phase I. Surprisingly, interferons suppressed reactivation only during a limited period early in phase I preceding productive virus growth. Sensitivity to type II interferon was selectively lost if viral ICP0, which normally accumulates later in phase I, was expressed before reactivation. Thus, interferons suppress reactivation by preventing initial expression of latent genomes but are ineffective once phase I viral proteins accumulate, limiting interferon action. This demonstrates that inducible reactivation from latency is only transiently sensitive to interferon. Moreover, it illustrates how latent pathogens escape host immune control to periodically replicate by rapidly deploying an interferon-resistant state.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the β-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention., Author summary Chikungunya virus (CHIKV) is a globally emerging virus that can cause significant disease, including a prolonged and painful arthritis. The virus is spread by mosquitoes that circulate in many regions of the world including the United States. Currently, there are no available vaccines or therapies to treat CHIKV infection. In this report, we identified and characterized a large panel of antibodies against CHIKV from two donors that contracted the viral infection in the Dominican Republic. These antibodies target a number of different regions of the membrane proteins that coat the surface of the virus, and many can inhibit the ability of CHIKV to infect cells. Two of the antibodies were shown to protect mice from a lethal dose of CHIKV. These antibodies have therapeutic potential, and provide insight into the human immune response that may facilitate vaccine development.

Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.

Hallucinogenic drugs, such as lysergic acid diethylamide (LSD), mescaline and psilocybin, alter perception and cognitive processes. All hallucinogenic drugs have in common a high affinity for the serotonin 5-HT(2A) receptor. Metabotropic glutamate 2/3 (mGlu2/3) receptor ligands show efficacy in modulating the cellular and behavioral responses induced by hallucinogenic drugs. Here, we explored the effect of chronic treatment with the mGlu2/3 receptor antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)-propionic acid (LY341495) on the hallucinogenic-like effects induced by LSD (0.24mg/kg). Mice were chronically (21 days) treated with LY341495 (1.5mg/kg), or vehicle, and experiments were carried out one day after the last injection. Chronic treatment with LY341495 down-regulated [(3)H]ketanserin binding in somatosensory cortex of wild-type, but not mGlu2 knockout (KO), mice. Head-twitch behavior, and expression of c-fos, egr-1 and egr-2, which are responses induced by hallucinogenic 5-HT(2A) agonists, were found to be significantly decreased by chronic treatment with LY341495. These findings suggest that repeated blockade of the mGlu2 receptor by LY341495 results in reduced 5-HT(2A) receptor-dependent hallucinogenic effects of LSD.

It has been suggested that severe adverse life events during pregnancy increase the risk of schizophrenia in the offspring. The serotonin 5-HT2Aand the metabotropic glutamate 2 (mGlu2) receptors both have been the target of considerable attention regarding schizophrenia and antipsychotic drug development. We tested the effects of maternal variable stress during pregnancy on expression and behavioral function of these two receptors in mice. Prenatal stress increased 5-HT2Aand decreased mGlu2 expression in frontal cortex, a brain region involved in perception, cognition, and mood. This pattern of expression of 5-HT2Aand mGlu2 receptors was consistent with behavioral alterations, including increased head-twitch response to the hallucinogenic 5-HT2Aagonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane] and decreased mGlu2-dependent antipsychotic-like effect of the mGlu2/3 agonist LY379268 (1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate) in adult, but not prepubertal, mice born to stressed mothers during pregnancy. Cross-fostering studies determined that these alterations were not attributable to effects of prenatal stress on maternal care. Additionally, a similar pattern of biochemical and behavioral changes were observed in mice born to mothers injected with polyinosinic:polycytidylic acid [poly(I:C)] during pregnancy as a model of prenatal immune activation. These data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for schizophrenia and other psychiatric disorders.

The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin–CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1. Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B. Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.

In schizophrenia patients, optimal treatment with antipsychotics requires weeks to months of sustained drug therapy. However, single administration of antipsychotic drugs can reverse schizophrenia-like behavioral alterations in rodent models of psychosis. This raises questions about the physiological relevance of such antipsychotic-like activity.This study evaluates the effects of chronic treatment with clozapine on the cellular and behavioral responses induced by the hallucinogenic serotonin 5-HT(2A) receptor agonist lysergic acid diethylamide (LSD) as a mouse model of psychosis.Mice were treated chronically (21 days) with 25 mg/kg/day clozapine. Experiments were conducted 1, 7, 14, and 21 days after the last clozapine administration. [(3)H]Ketanserin binding and 5-HT ( 2A ) mRNA expression were determined in mouse somatosensory cortex. Head-twitch behavior, expression of c-fos, which is induced by all 5-HT(2A) agonists, and expression of egr-1 and egr-2, which are LSD-like specific, were assayed.Head-twitch response was decreased and [(3)H]ketanserin binding was downregulated in 1, 7, and 14 days after chronic clozapine. 5-HT ( 2A ) mRNA was reduced 1 day after chronic clozapine. Induction of c-fos, but not egr-1 and egr-2, was rescued 7 days after chronic clozapine. These effects were not observed after short treatment (2 days) with clozapine or chronic haloperidol (1 mg/kg/day).Our findings provide a murine model of chronic atypical antipsychotic drug action and suggest downregulation of the 5-HT(2A) receptor as a potential mechanism involved in these persistent therapeutic-like effects.

Cancer cells often over-express specific genes as a result of translocation or gene amplification. We developed a high throughput screening protocol to mimic cancer-related over-expression in Saccharomyces cerevisiae deletion strain libraries to identify genes whose functions become essential only when the cancer-related query gene is over-expressed. The resulting synthetic dosage lethal (SDL) interactions uncover conserved genes that can be targeted to kill cancer cells but leave normal cells unaffected, providing a cancer-specific therapy. The CKS1b gene is located on chromosome 1q21, a region that is frequently amplified in breast, lung and liver cancers. Cks1b is a conserved regulatory subunit of cyclin-CDK complexes which functions during cell cycle progression. The SDL screen identified multiple pathways affecting mitotic progression that are essential when yeast CKS1 is over-expressed. One pathway controls a morphological checkpoint that leads to stabilization of the mitotic inhibitor Swe1 (Wee1 in mammals) resulting in delayed mitosis when cell polarity is disrupted. Swe1, a tyrosine kinase, inhibits the cyclin dependent kinase (CDK) complex by phosphorylation of a conserved tyrosine-19 on the yeast CDK, Cdc28. We find that the SDL interaction between CKS1 and the morphological checkpoint requires both Swe1 and the Cdc28 tyrosine-19 residue. Normal turnover of Swe1 occurs via SCF-mediated degradation after phosphorylation by the cyclin-CDK complex and the Polo-like kinase (Cdc5). Since, mutant alleles of CDC5 were identified in the SDL screen, we investigated the effect of targeting human PLK1, the mammalian Cdc5 ortholog, in breast cancers with varying expression of Cks1b. We first correlated RNAi knock down of PLK1 across 28 breast cancer cell lines [1] with Cks1b expression data taken from the Cancer Cell Line Encyclopedia [2]. Importantly, we find that growth inhibition by PLK1 knockdown correlates with increased Cks1b levels (r = -0.554, top 0.8th percentile). TCGA data also provides evidence for this SDL in breast cancer, since CKS1b over-expression and down-regulation of PLK1 expression rarely co-occur (mutual exclusion p-value 6.47x10-8). Together, these analyses support the hypothesis that PLK1 activity is essential when CKS1b levels are elevated. Finally, using shRNAs, we independently confirmed the PLK1 knockdown sensitivity in 8 breast cancer cell lines. Three of the 4 sensitive lines show increased CKS1b expression, while 3 of the 4 insensitive lines show low CKS1b expression, corroborating the correlation of CKS1b and PLK1 levels and lending support to conservation of the genetic interaction. [1] Marcotte, et al., Cancer Discovery, 2:172 (2012) [2] Barretina, et al., Nature, 483:603 (2012) Citation Format: Robert J. D. Reid, Xing Du, John C. DIttmar, Vinayak Rayannavar, Ivana Sunjevaric, Matthew Maurer, Rodney Rothstein. A synthetic genetic interaction screen in yeast identifies Plk1 as a promising therapeutic target in cancer cells that overexpress Cks1b. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2014-5088