Discovery and Synthesis of Heterobifunctional Degraders of Rearranged during Transfection (RET) Kinase.

We describe the design, synthesis, and structure-activity relationship (SAR) of heterobifunctional RET ligand-directed degraders (LDDs) derived from three different second-generation RET inhibitors. These LDDs are composed of a target binding motif (TBM) that binds to the RET protein, a linker, and a cereblon binding motif (CBM) as the E3 ligase recognition unit. This led to the identification of a series of pyrazolopyridine-based heterobifunctional LDDs, as exemplified by compound .

Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration.

Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a hostile tumor microenvironment that renders T cells dysfunctional. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment.

DGKα/ζ inhibition lowers the TCR affinity threshold and potentiates antitumor immunity.

Diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG) signaling by converting DAG to phosphatidic acid, thereby suppressing pathways downstream of T cell receptor signaling. Using a dual DGKα/ζ inhibitor (DGKi), tumor-specific CD8 T cells with different affinities (TRP1 and TRP1), and altered peptide ligands, we demonstrate that inhibition of DGKα/ζ can lower the signaling threshold for T cell priming. TRP1 and TRP1 CD8 T cells produced more effector cytokines in the presence of cognate antigen and DGKi.

DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses.

Discovery of Potent, Dual-Inhibitors of Diacylglycerol Kinases Alpha and Zeta Guided by Phenotypic Optimization.

We describe a phenotypic screening and optimization strategy to discover compounds that block intracellular checkpoint signaling in T-cells. We identified dual DGKα and ζ inhibitors notwithstanding the modest similarity between α and ζ relative to other DGK isoforms. Optimized compounds produced cytokine release and T-cell proliferation consistent with DGK inhibition and potentiated an immune response in human and mouse T-cells.

Prospective prediction of plasma pharmacokinetics of a novel immune-modulating agent in cancer patients after intra-tumoral administration: translation from non-clinical species to humans.

Intra-tumoral (I-TUMOUR) delivery is being widely explored for novel anti-cancer agents. This route is anticipated to result in high tumour concentrations leading to better efficacy and safety. Prediction of human systemic pharmacokinetics (PK) from non-clinical species facilitates understanding of pharmacokinetic-pharmacodynamic relationships, efficient dose selection, and risk assessment of novel drugs.

DGKζ exerts greater control than DGKα over CD8 T cell activity and tumor inhibition.

Two isoforms of diacylglycerol kinases (DGKs), DGKα and DGKζ, are primarily responsible for terminating DAG-mediated activation of Ras and PKCθ pathways in T cells. A direct comparison of tumor growth between mice lacking each isoform has not been undertaken. We evaluated the growth of three syngeneic tumor cell lines in mice lacking either DGKα or DGKζ in the presence or absence of treatment with anti-PD1 and determined that (i) mice deficient in DGKζ conferred enhanced control of tumor relative to mice deficient in DGKα and (ii) deficiency of DGKζ acted additively with anti-PD1 in tumor control.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors.

Interrogating the immune-modulating roles of radiation therapy for a rational combination with immune-checkpoint inhibitors in treating pancreatic cancer.

Background: Radiation therapy (RT) has the potential to enhance the efficacy of immunotherapy, such as checkpoint inhibitors, which has dramatically altered the landscape of treatments for many cancers, but not yet for pancreatic ductal adenocarcinoma (PDAC). Our prior studies demonstrated that PD ligand-1 and indoleamine 2,3-dioxygenase 1 (IDO1) were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT, suggesting RT may prime PDAC for PD-1 blockade antibody (αPD-1) or IDO1 inhibitor (IDO1i) treatments. In this study, we investigated the antitumor efficacy of the combination therapies with radiation and PD-1 blockade or IDO1 inhibition or both.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination.

Safety and efficacy of anti-PD-L1 therapy in the woodchuck model of HBV infection.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters.

High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening).

A novel small molecule inhibitor of hepatitis C virus entry.

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations.

Ultrasensitive genotypic detection of antiviral resistance in hepatitis B virus clinical isolates.

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population.

Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naïve patients is rare through 5 years of therapy.

Unlabelled: Patients with chronic hepatitis B virus (HBV) infection who develop antiviral resistance lose benefits of therapy and may be predisposed to further resistance. Entecavir (ETV) resistance (ETVr) results from HBV reverse transcriptase substitutions at positions T184, S202, or M250, which emerge in the presence of lamivudine (LVD) resistance substitutions M204I/V +/- L180M. Here, we summarize results from comprehensive resistance monitoring of patients with HBV who were continuously treated with ETV for up to 5 years.

Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies.

APOBEC3G is an antiviral host factor capable of inhibiting the replication of both exogenous and endogenous retroviruses as well as hepatitis B, a DNA virus that replicates through an RNA intermediate. To gain insight into the mechanism whereby APOBEC3G restricts retroviral replication, we investigated the subcellular localization of the protein. Herein, we report that APOBEC3G localizes to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of nontranslating mRNAs.

Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization.

To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23.

Biosynthesis of glycosylphosphatidylinositol is essential to the survival of the protozoan parasite Toxoplasma gondii.

The PIGA gene from Toxoplasma gondii has been cloned and characterized. Like mammalian PIGA, the transmembrane and C-terminal domains are sufficient to direct localization to the parasite endoplasmic reticulum. A functional copy of PIGA is required for tachyzoite viability, demonstrating that glycosylphosphatidylinositol biosynthesis is an essential process in T.

Clostridium septicum alpha-toxin is active against the parasitic protozoan Toxoplasma gondii and targets members of the SAG family of glycosylphosphatidylinositol-anchored surface proteins.

As is the case with many other protozoan parasites, glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of Toxoplasma gondii tachyzoites. The mechanisms by which T. gondii GPI-anchored proteins are synthesized and transported through the unusual triple-membrane structure of the parasite pellicle to the plasma membrane remain largely unknown.