Reverse Translating Molecular Determinants of Anti-Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models.

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Discovery of MK-4688: an Efficient Inhibitor of the HDM2-p53 Protein-Protein Interaction.

Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties. Ultimately, a strategy focused on leveraging known binding hot spots coupled with biostructural information to guide the design of conformationally constrained analogs and a focus on efficiency metrics led to the discovery of MK-4688 (compound 56), a highly potent, selective, and low-molecular-weight inhibitor suitable for clinical investigation.

Corrigendum: Effective Anti-tumor Response by TIGIT Blockade Associated With FcγR Engagement and Myeloid Cell Activation.

[This corrects the article DOI: 10.3389/fimmu.2020.573405.].

Effective Anti-tumor Response by TIGIT Blockade Associated With FcγR Engagement and Myeloid Cell Activation.

The molecule "T cell immunoreceptor with immunoglobulin and ITIM domain," or TIGIT, has recently received much attention as a promising target in the treatment of various malignancies. In spite of the quick progression of anti-TIGIT antibodies into clinical testing both as monotherapy and in combination with programmed cell death-1 (PD-1)-directed immune checkpoint blockade, the molecular mechanism behind the observed therapeutic benefits remains poorly understood. Here we demonstrate, using mouse tumor models, that TIGIT blocking antibodies with functional Fc binding potential induce effective anti-tumor response. Our observations reveal that the anti-TIGIT therapeutic effect is not achieved by depletion of intratumoral regulatory T cells (Treg) or any cell population expressing TIGIT, but instead is mediated by possible "reverse activating signals" through FcγRs on myeloid cells, inducing expression of various mediators such as cytokines and chemokines. Furthermore, we discovered an induction of a robust and persistent granzyme B and perforin response, distinct from a predominantly interferon-γ (IFN-γ)-driven anti-PD-1 blockade. Our observations, for the first time, provide the basis for a mechanistic hypothesis involving the requirement of a functional Fc domain of anti-TIGIT monoclonal antibodies, of which various isotypes are currently under intense clinical investigation.

Dinaciclib induces immunogenic cell death and enhances anti-PD1-mediated tumor suppression.

Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.

Mechanistically probing lipid-siRNA nanoparticle-associated toxicities identifies Jak inhibitors effective in mitigating multifaceted toxic responses.

A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.

Noninvasive imaging of lipid nanoparticle-mediated systemic delivery of small-interfering RNA to the liver.

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.

The crosslinking and antimicrobial properties of tunichrome.

Tunichromes are small peptides containing one or more dehydrodopa derived units that have been identified in the blood cells of at least eleven species of tunicates. Incubation of tunichromes isolated from Ascidia nigra hemocytes (or model dopa-containing compounds) under oxidative conditions with either lysozyme, cytochrome c or ovalbumin resulted in a time-dependent polymerization of these test proteins to dimers, trimers, tetramers and potentially to other oligomers. These results indicate that the oxidation products of tunichromes possess inherent crosslinking properties. Hence it is possible that tunichromes participate in tunic production by forming adducts and crosslinks with structural proteins and/or carbohydrate polymers, similar to the well-understood process of insect cuticle hardening. Since such crosslinking potentials could also be beneficial for defense reactions against invading microorganisms, antibacterial activity of tunichromes was tested using both a radial diffusion assay and the Microtox(R) test. Tunichromes exhibited antimicrobial activity against gram-negative bacteria Escherichia coli and Photobacterium phosphorium. However, they did not show any antimicrobial activity against the gram-positive bacteria Staphylococcus aureus at the concentrations tested. We propose that the crosslinking and antimicrobial functions are both based on the reactivity of dehydrodopa units present in the tunichromes, and their subsequent ability to form highly reactive quinone methides.

The C-terminal kinase domain of the p34cdc2-related PITSLRE protein kinase (p110C) associates with p21-activated kinase 1 and inhibits its activity during anoikis.

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

Involvement of beta 1,4 galactosyltransferase 1 and Gal beta1-->4GlcNAc groups in human hepatocarcinoma cell apoptosis.

Beta 1,4 galactosyltransferase 1 (beta 1,4GT1) synthesizes Gal beta 1-->4GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of beta 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal beta 1-->4GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with beta 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of beta 1,4GT1, which also led to the increased Gal beta 1-->4GlcNAc groups on the transfected cell surface. All the observations suggested that beta 1,4GT1 and Gal beta 1-->4GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.

Interaction of p58(PITSLRE), a G2/M-specific protein kinase, with cyclin D3.

The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.