Alex Braiman

(1) Background: Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide. The tumor microenvironment (TME) is a key driver of disease progression, shaping both pro- and anti-tumor immune responses. Interleukin-1β (IL-1β) is a pleiotropic pro-inflammatory cytokine that participates in both homeostatic and pathological conditions. It has been shown to be upregulated in many tumors, including CRC, where it promotes angiogenesis, immunosuppression, and resistance to chemotherapy. (2) Objective: Elucidate the correlation between TME IL-1β and colon microbiotic changes during CRC progression and approach inhibition of IL-1β as a therapeutic strategy in combination with chemotherapy. (3) Design: Using an orthotopic mouse model of CRC, we compared tumor growth, metastatic spread, immune infiltration and microbiome composition in control and IL-1β knockout mice. In addition, we studied the effects of IL-1β neutralization alone or in combination with FOLFOX chemotherapy on CRC prognosis. (4) Results: IL-1β promoted CRC progression by enhancing pro-inflammatory signalling, recruiting immunosuppressive myeloid cells, and driving microbiome dysbiosis. In contrast, IL-1β deficiency increased antigen-presenting myeloid cells and CD⁸⁺ T-cell infiltration, leading to delayed tumor growth and reduced metastasis. FOLFOX chemotherapy alone exacerbated systemic and local inflammation, facilitating liver metastasis. Strikingly, the addition of IL-1β blockade abrogated FOLFOX-induced inflammation, enhanced anti-tumor immunity, and prevented metastatic spread. (5) Conclusions: IL-1β from the TME links microbiome dysbiosis, tumor-promoting inflammation and poor chemotherapy outcomes in CRC. Combining IL-1β inhibition with standard FOLFOX chemotherapy represents a promising therapeutic approach to improve prognosis in CRC patients.

Protein kinase C θ (PKCθ) is expressed in T lymphocytes, in which it plays a crucial role in cell activation, proliferation, and differentiation. Previously, we discovered that the Thr ³³⁵ ‐Pro motif within the PKCθ‐V3 regulatory domain serves as a critical site for PKCθ activation and demonstrated that phosphorylation of the Thr ³³⁵ ‐Pro motif creates a binding site for the peptidyl‐prolyl cis‐trans isomerase (PPIase), Pin1. Herein, we elaborate on the functional consequences of the Pin1‐PKCθ association and identify Pin1 as a regulator of PKCθ catalytic activity. Pin1 downregulated the activity of PKCθ in vitro in PMA‐stimulated human Jurkat T cells and C57BL/6J mouse spleen‐ and thymus‐derived T lymphocytes, an effect that was reversed by juglone, a Pin1‐activity inhibitor. Utilizing Pin1 isomerase‐deficient mutants, we demonstrate that the functionality of the Pin1 catalytic domain is essential for its regulation of PKCθ, suggesting that Pin1 mediates its inhibitory effect on PKCθ via cis‐trans isomerization. In silico docking analysis supported the role of critical residues within the Pin1‐PPIase domain that enables the cis‐trans interconversion of the PKCθ phospho‐Thr ³³⁵ ‐Pro motif. Retroviral knockdown of Pin1 in Jurkat T cells led to an elevation in PKCθ kinase activity. Furthermore, stimulation of [Lck ^cre × Pin1 ^lox ] F ₁ mice‐derived Pin1‐deficient T cells augmented the phosphorylation of SPAK kinase, a bona fide PKCθ downstream substrate. Together, our results support a role for the Pin1 isomerase as a regulator of PKCθ activity and highlight the potential contribution of Pin1 to the fine‐tuning of the T cell activation dynamics.

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3⁺CD8⁺ and NKp46⁺cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.

Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient’s fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient’s fibroblasts. Finally, the patient’s fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart.

Over 50% of human papilloma positive head-and-neck cancer (HNC^HPV+) patients harbor genomic-alterations in PIK3CA, leading to hyperactivation of the phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway. Nevertheless, despite PI3K pathway activation in HNC^HPV+ tumors, the anti-tumor activities of PI3K pathway inhibitors are moderate, mostly due to the emergence of resistance. Thus, for potent and long-term tumor management, drugs blocking resistance mechanisms should be combined with PI3K inhibitors. Here, we delineate the molecular mechanisms of the acquisition of resistance to two isoform-selective inhibitors of PI3K (isiPI3K), alpelisib (BYL719) and taselisib (GDC0032), in HNC^HPV+ cell lines. By comparing the transcriptional landscape of isiPI3K-sensitive tumor cells with that of their corresponding isiPI3K-acquired-resistant tumor cells, we found upregulation of insulin growth factor 2 (IGF2) in the resistant cells. Mechanistically, we show that upon isiPI3K treatment, isiPI3K-sensitive tumor cells upregulate the expression of IGF2 to induce cell proliferation via the activation of the IGF1 receptor (IGF1R). Stimulating tumor cells with recombinant IGF2 limited isiPI3K efficacy and released treated cells from S phase arrest. Knocking-down IGF2 with siRNA, or blocking IGF1R with AEW541, resulted in superior anti-tumor activity of isiPI3K in vitro and ex vivo. In vivo, the combination of isiPI3K and IGF1R inhibitor induced stable disease in mice bearing either tumors generated by the HNC^HPV+ UM-SCC47 cell line or HPV+ patient-derived xenografts. These findings indicate that IGF2 and the IGF2/IGF1R pathway may constitute new targets for combination therapies to enhance the efficacy of PI3K inhibitors for the treatment of HNC^HPV+.

Background: The study of thePlasmodium falciparumheavy metal transporter genepfmdr2employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium¹⁰⁹which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in theP. falciparumparasite is proposed as an alternative to the use of radioactive labelled heavy metals.Methods: Plasmodium falciparumCd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl₂concentrations and for different time periods followed by staining of the infected red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy.Results: The results show that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM fluorescence levels were negligible and increased only at high concentrations of Cd and at long incubation periods, but to a much lesser extent than the sensitive strain.No Cd uptake is observed in uninfected red blood cells populations originating from cultures infected with either sensitive or resistant strain. In addition, confocal microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates in the parasite itself.Conclusions: The dual fluorescent labelling is a valid method for detecting heavy metal accumulation inP. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent labelling enables us to differentiate between the different populations existing in aP. falciparuminfected red blood cells cultures and thus actually study a phenomenon at the level of a single cell.

Recent studies have uncovered the links between aging, rejuvenation and polar protein transport in the budding yeast Saccharomyces cerevisiae. Here, we examined a still unexplored possibility for co-regulation of polar mRNA transport and lifespan. To monitor the amount and distribution of mRNA-containing granules in mother and daughter cells, we used a fluorescent mRNA-labeling system, with MFA2 as a reporter gene. The results obtained showed that deletion of the selected longevity regulators in budding yeast had a significant impact on the polar mRNA transport. This included changes in the amount of mRNA-containing granules in cytoplasm, their aggregation and distribution between the mother and daughter cells. A significant negative correlation was found between strain-specific longevity, amount of granules and total fluorescent intensity both in mother and daughter cells. As indicated by the coefficient of determination, approximately 50–75% of variation in yeast lifespan could be attributed to the differences in polar mRNA transport.