Сytotoxic effect of CAR-T cells against modified MCF-7 breast cancer cell line.

The most often diagnosed and fatal malignancy in women is breast cancer. The International Agency for Research on Cancer (IARC) estimates that there are 2.26 million new cases of cancer in 2020. Adoptive cell therapy using T cells with chimeric antigen receptor shows potential for the treatment of solid tumors, such as breast cancer. In this work the effectiveness of CAR-T cells against monolayer and three-dimensional bioprinted tumor-like structures made of modified MCF-7 breast cancer cells was assessed. The cytokine profile of supernatants after co-cultivation of MCF-7 tumor cell models with CAR-T cells was also measured to reveal the inflammatory background associated with this interaction.

Evaluation of CAR-T Cells' Cytotoxicity against Modified Solid Tumor Cell Lines.

In recent years, adoptive cell therapy has gained a new perspective of application due to the development of technologies and the successful clinical use of CAR-T cells for the treatment of patients with malignant B-cell neoplasms. However, the efficacy of CAR-T therapy against solid tumor remains a major scientific and clinical challenge. In this work, we evaluated the cytotoxicity of 2nd generation CAR-T cells against modified solid tumors cell lines-lung adenocarcinoma cell line H522, prostate carcinoma PC-3M, breast carcinoma MDA-MB-231, and epidermoid carcinoma A431 cell lines transduced with lentiviruses encoding red fluorescent protein Katushka2S and the CD19 antigen. A correlation was demonstrated between an increase in the secretion of proinflammatory cytokines and a decrease in the confluence of tumor cells' monolayer. The proposed approach can potentially be applied to preliminarily assess CAR-T cell efficacy for the treatment of solid tumors and estimate the risks of developing cytokine release syndrome.

New Evidence of the Importance of Weak Interactions in the Formation of PML-Bodies.

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4-6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML-/-), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid-liquid phase separation (LLPS) leading to the formation of PML bodies.

The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics.

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of "small" (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as "seeds" of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

Inhibition of the DNA damage response phosphatase PPM1D reprograms neutrophils to enhance anti-tumor immune responses.

PPM1D/Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human solid tumors. Recent reports associate gain-of-function mutations of PPM1D in immune cells with worse outcomes for several human cancers. Here we show that mice with genetic knockout of Ppm1d or with conditional knockout of Ppm1d in the hematopoietic system, in myeloid cells, or in neutrophils all display significantly reduced growth of syngeneic melanoma or lung carcinoma tumors. Ppm1d knockout neutrophils infiltrate tumors extensively. Chemical inhibition of Wip1 in human or mouse neutrophils increases anti-tumor phenotypes, p53-dependent expression of co-stimulatory ligands, and proliferation of co-cultured cytotoxic T cells. These results suggest that inhibition of Wip1 in neutrophils enhances immune anti-tumor responses.

Resveratrol enhances pluripotency of mouse embryonic stem cells by activating AMPK/Ulk1 pathway.

Resveratrol, a natural polyphenolic compound, shows many beneficial effects in various animal models. It increases efficiency of somatic cell reprograming into iPSCs and contributes to cell differentiation. Here, we studied the effect of resveratrol on proliferation and pluripotency of mouse embryonic stem cells (mESCs). Our results demonstrate that resveratrol induces autophagy in mESCs that is provided by the activation of the AMPK/Ulk1 pathway and the concomitant suppression of the activity of the mTORC1 signaling cascade. These events correlate with the enhanced expression of pluripotency markers Oct3/4, Sox2, Nanog, Klf4, SSEA-1 and alkaline phosphatase. Pluripotency is retained under resveratrol-caused retardation of cell proliferation. Given that the Ulk1 overexpression enhances pluripotency of mESCs, the available data evidence that mTOR/Ulk1/AMPK-autophagy network provides the resveratrol-mediated regulation of mESC pluripotency. The capability of resveratrol to support the mESC pluripotency provides a new approach for developing a defined medium for ESC culturing as well as for better understanding signaling events that govern self-renewal and pluripotency.

VEGFR2-specific FnCAR effectively redirects the cytotoxic activity of T cells and YT NK cells.

T and NK cells armed with chimeric antigen receptors (CAR) are promising tools for the specific elimination of cancer cells. In most CAR designs implemented to date, the recognition of target cells is mediated by single-chain variable fragments (scFvs) derived from murine monoclonal antibodies. This format, however, has a number of limitations, including its relatively large size and potential immunogenicity in humans. In this study, we explored the feasibility of using human fibronectin type III domains (Fn3) as the antigen recognition domain in CARs. Human Fn3 domains have lower predicted immunogenicity compared to mouse-derived sequences, and a reduced molecular weight compared to scFvs. We created a functional CAR using a VEGFR2-specific Fn3 module replacing the conventional scFv. The resulting FnCAR specifically potentiates the cytotoxic activity of human T cells and YT NK cells in the presence of VEGFR2-positive targets. These findings demonstrate that Fn3 domains can be used in CARs for antigen recognition.

Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors.

A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.