Cancer immunotherapy with enveloped self-amplifying mRNA CARG-2020 that modulates IL-12, IL-17 and PD-L1 pathways to prevent tumor recurrence.

Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

The mTOR Signaling Pathway Interacts with the ER Stress Response and the Unfolded Protein Response in Cancer.

The mTOR complex 1 (mTORC1) coordinates several important environmental and intracellular cues to control a variety of biological processes, such as cell growth, survival, autophagy, and metabolism, in response to energy levels, growth signals, and nutrients. The endoplasmic reticulum (ER) is a crucial intracellular organelle that is essential for numerous cellular functions, including the synthesis, folding, and modification of newly synthesized proteins, stress responsiveness, and maintainence of cellular homeostasis. mTOR-mediated upregulation of protein synthesis induces the accumulation of misfolded or unfolded proteins in the ER lumen, which induces ER stress, leading to activation of the unfolded protein response (UPR) pathway. Reciprocally, ER stress regulates the PI3K/AKT/mTOR signaling pathway. Therefore, under pathologic conditions, the cross-talk between the mTOR and UPR signaling pathways during cellular stress can critically affect cancer cell fate and may be involved in the pathogenesis and therapeutic outcome of cancer. Here, we discuss accumulating evidence showing the mechanism of action, interconnections, and molecular links between mTOR signaling and ER stress in tumorigenesis and highlights potential therapeutic implications for numerous cancers.

High glucose promotes regulatory T cell differentiation.

The consumption of processed foods and sugary sodas in Western diets correlates with an increased incidence of obesity, metabolic syndromes such as type 2 diabetes, cardiovascular diseases, and autoimmune diseases including inflammatory bowel disease and rheumatoid arthritis. All these diseases have an inflammatory component, of which T lymphocytes can play a critical role in driving. Much has been learned regarding the importance of sugar, particularly glucose, in fueling effector versus regulatory T cells that can promote or dampen inflammation, respectively. In particular, glucose and its metabolic breakdown products via glycolysis are essential for effector T cell differentiation and function, while fatty acid-fueled oxidative phosphorylation supports homeostasis and function of regulatory T cells. Nevertheless, a critical knowledge gap, given the prevalence of diabetes in Western societies, is the impact of elevated glucose concentrations on the balance between effector versus regulatory T cells. To begin addressing this, we cultured naïve CD4+ T cells with different concentrations of glucose, and examined their differentiation into effector versus regulatory lineages. Surprisingly, high glucose promoted regulatory T cell differentiation and inhibited Th1 effector differentiation. This skewing towards the regulatory lineage occurred via an indirect mechanism that depends on lactate produced by activated glycolytic T cells. Addition of lactate to the T cell differentiation process promotes the differentiation of Treg cells, and activates Akt/mTOR signaling cascade. Hence, our findings suggest the existence of a novel feedback mechanism in which lactate produced by activated, differentiating T cells skews their lineage commitment towards the regulatory fate.

Interleukin-17 Family Cytokines in Metabolic Disorders and Cancer.

Interleukin-17 (IL-17) family cytokines are potent drivers of inflammatory responses. Although IL-17 was originally identified as a cytokine that induces protective effects against bacterial and fungal infections, IL-17 can also promote chronic inflammation in a number of autoimmune diseases. Research in the last decade has also elucidated critical roles of IL-17 during cancer development and treatment. Intriguingly, IL-17 seems to play a role in the risk of cancers that are associated with metabolic disorders. In this review, we summarize our current knowledge on the biochemical basis of IL-17 signaling, IL-17's involvement in cancers and metabolic disorders, and postulate how IL-17 family cytokines may serve as a bridge between these two types of diseases.

ß-Alkyloxazolochlorins: Revisiting the Ozonation of Octaalkylporphyrins, and Beyond.

The reaction of ß-octaalkylporphyrins (octaethylporphyrin and etioporphyrin I) with ozone generated the corresponding heptaalkyloxazolochlorinhemiacetals in which a pyrrolic subunit of the porphyrins was replaced by an oxazoline moiety. Thus, a pyrrolic ß-carbon with its alkyl substituent was excised and replaced by an oxygen atom, and the neighboring ß-carbon was hydroxylated. This work clarifies the nature of the products first described by Fischer and Dezelic, in 1933, and verifies the work by Shulg'a and coworkers, from 1977. Furthermore, the chemistry of the oxazolochlorin hemiacetals was studied: They could be dehydroxylated or converted to alkyl acetals and gem-dialkyl derivatives, all possessing chlorin-type optical spectra. Their oxidative conversions generated a unique tetrahydrofuran-linked oxazolochlorin dimer and a hexaethylporpholactone. The work expands on the knowledge of converting porphyrins to porphyrinoids of potential utility containing nonpyrrolic building heterocycles.

Octaethyl-1,3-oxazinochlorin: A ß-Octaethylchlorin Analogue Made by Pyrrole Expansion.

Treatment of the oxime of octaethyloxochlorin 4, available from octaethylporphyrin 3, under Beckmann conditions provided not the expected lactam, but octaethyl-1,3-oxazinochlorin 8, in which a pyrrole moiety of the parent oxochlorin was expanded by an oxygen atom to an 1,3-oxazinone moiety. Its mechanism of formation was demonstrated to occur along an "abnormal Beckmann" pathway, followed by intramolecular ring closure and hydrolysis. The work expands the methodologies known to convert octaethylporphyrin to pyrrole-modified porphyrin analogues.

Oxazolochlorins. 9. meso-Tetraphenyl-2-oxabacteriochlorins and meso-tetraphenyl-2,12/13-dioxabacteriochlorins.

The formal replacement of one or two pyrrole groups in meso-tetraphenylporphyrin by oxazole moieties is described, generating inter alia the bacteriochlorin-type chromophores oxazolobacteriochlorins (oxabacteriochlorins) and bisoxazolobacteriochlorins (dioxabacteriochlorins). The key step is the conversion of a ß,ß'-dihydroxy-functionalized pyrroline group into an oxazolone or (substituted) oxazole. Depending on the substitution pattern on the oxazole or oxazoline moieties, mono- and dioxabacteriochlorins may have chlorin- or bacteriochlorin-like spectra. The optical properties (as measured by UV-vis and fluorescence spectroscopies) of the novel oxa- and dioxabacteriochlorins are described and contrasted against benchmark chlorins and bacteriochlorins. The conformations of a representative number of mono- and dioxabacteriochlorins, as their free bases or Zn(II) complexes, were determined by single-crystal X-ray diffractometry. They proved to be essentially planar, showing that the modulation of their optical properties is primarily due to their intrinsic electronic structures and electronic substituent effects and are not largely affected by conformational effects. The mono- and bisoxazolobacteriochlorins are a novel class of readily prepared and oxidatively stable chlorin and bacteriochlorin analogues with tunable optical spectra that, in part, reach into the NIR.

meso-Aryl-3-alkyl-2-oxachlorins.

The formal replacement of a pyrrole moiety of meso-tetraarylporphyrin 1 by an oxazole moiety is described. The key step is the conversion of porpholactones 4 (prepared by a known two-step oxidation procedure from 1) by addition of alkyl Grignard reagent to form meso-tetraaryl-3-alkyl-2-oxachlorins 9 (alkyloxazolochlorins; alkyl = Me, Et, iPr). Hemiacetal 9 can be converted to an acetal, reduced to an ether, or converted to bis-alkyloxazolochlorins 11. The optical properties (UV-visible and fluorescence spectroscopy) are described. The chlorin-like optical properties of the alkyloxazolochlorins are compared to regular chlorins, such as 2,3-dihydroxychlorins and nonalkylated oxazolochlorins made by reduction from porpholactone 4. The conformations of the mono- and bis-alkylated 2-oxachlorins, as determined by single crystal X-ray diffractometry, are essentially planar, thus proving that their optical properties are largely due to their intrinsic electronic properties and not affected by conformational effects. The mono- and bis-3-alkyl-2-oxachlorins are a class of readily prepared and oxidatively stable chlorins.