Pegylated-liposomes increase the efficacy of Idelalisib in lymphoma B-cells.

New drugs and technologies are continuously developed to improve the efficacy and minimize the critical side effects of cancer treatments. The present investigation focuses on the development of a liposomal formulation for Idelalisib, a small-molecule kinase inhibitor approved for the treatment of lymphoid malignancies. Idelalisib is a potent and selective antitumor agent, but it is not indicated nor recommended for first-line treatment due to fatal and serious toxicities. Herein, liposomes are proposed as a delivery tool to improve the therapeutic profile of Idelalisib. Specifically, PEGylated liposomes were prepared, and their physicochemical and technological features were investigated. Light-scattering spectroscopy and cryo-transmission electron microscopy revealed nanosized unilamellar vesicles, which were proved to be stable in storage and in simulated biological fluids. The cytotoxicity of the liposome formulation was investigated in a human non-Hodgkin's lymphoma B cell line. Idelalisib was able to induce death of tumor cells if delivered by the nanocarrier system at increased efficacy. These findings suggest that combining Idelalisib and nanotechnologies may be a powerful strategy to increase the antitumor efficacy of the drug.

Tumor Microenvironment Landscapes Supporting EGFR-mutant NSCLC Are Modulated at the Single-cell Interaction Level by Unesbulin Treatment.

Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs. SIGNIFICANCE: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements.

EGFR signaling pathway as therapeutic target in human cancers.

Epidermal Growth Factor Receptor (EGFR) enacts major roles in the maintenance of epithelial tissues. However, when EGFR signaling is altered, it becomes the grand orchestrator of epithelial transformation, and hence one of the most world-wide studied tyrosine kinase receptors involved in neoplasia, in several tissues. In the last decades, EGFR-targeted therapies shaped the new era of precision-oncology. Despite major advances, the dream of converting solid tumors into a chronic disease is still unfulfilled, and long-term remission eludes us. Studies investigating the function of this protein in solid malignancies have revealed numerous ways how tumor cells dysregulate EGFR function. Starting from preclinical models (cell lines, organoids, murine models) and validating in clinical specimens, EGFR-related oncogenic pathways, mechanisms of resistance, and novel avenues to inhibit tumor growth and metastatic spread enriching the therapeutic portfolios, were identified. Focusing on non-small cell lung cancer (NSCLC), where EGFR mutations are major players in the adenocarcinoma subtype, we will go over the most relevant discoveries that led us to understand EGFR and beyond, and highlight how they revolutionized cancer treatment by expanding the therapeutic arsenal at our disposal.

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.

Fluorescence imaging of biochemical relationship between ubiquitinated histone 2A and Polycomb complex protein BMI1.

Several in vitro experiments have highlighted that the Polycomb group protein BMI1 plays a pivotal role in determining the biological functions of the Polycomb Repressor Complex 1 (PRC1), including its E3-ligase activity towards the Lys119 of histone H2A to yield ubiquitinated uH2A. The role of BMI1 in the epigenetic activity of PRC1 is particularly relevant in several cancers, particularly Non-Small Cell Lung Cancer (NSCLC). In this study, using indirect immunofluorescence protocols implemented on a confocal microscopy apparatus, we investigated the relationship between BMI1 and uH2A at different resolutions, in cultured (A549) and clinical NSCLC tissues, at the single cell level. In both cases, we observed a linear dependence of uH2A concentration upon BMI1 expression at the single nucleus level, indicating that the association of BMI1 to PRC1, which is needed for E3-ligase activity, occurs linearly in the physiological BMI1 concentration range. Additionally, in the NSCLC cell line model, a minor pool of uH2A may exist in absence of concurrent BMI1 expression, indicating non-exclusive, although predominant, role of BMI1 in the amplification of the E3-ligase activity of PRC1. A pharmacological downregulator of BMI1, PTC-209, was also tested in this context. Finally, the absence of significant colocalization (as measured by the Pearson's coefficient) between BMI1 and uH2A submicron clusters hints to a dynamic model where PRC1 resides transiently at ubiquitination sites. Beside unveiling subtle functional relationships between BMI1 and uH2A, these results also validate the use of uH2A as downstream "reporter" for BMI1 activity at the nuclear level in NSCLC contexts.

The Role of Prep1 in the Regulation of Mesenchymal Stromal Cells.

Molecular mechanisms governing cell fate decision events in bone marrow mesenchymal stromal cells (MSC) are still poorly understood. Herein, we investigated the homeobox gene Prep1 as a candidate regulatory molecule, by adopting Prep1 hypomorphic mice as a model to investigate the effects of Prep1 downregulation, using in vitro and in vivo assays, including the innovative single cell RNA sequencing technology. Taken together, our findings indicate that low levels of Prep1 are associated to enhanced adipogenesis and a concomitant reduced osteogenesis in the bone marrow, suggesting Prep1 as a potential regulator of the adipo-osteogenic differentiation of mesenchymal stromal cells. Furthermore, our data suggest that in vivo decreased Prep1 gene dosage favors a pro-adipogenic phenotype and induces a "browning" effect in all fat tissues.

Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.

Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients' blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.

Prep1 prevents premature adipogenesis of mesenchymal progenitors.

Transcriptional regulators are crucial in adipocyte differentiation. We now show that the homeodomain-containing transcription factor Prep1 is a repressor of adipogenic differentiation since its down-regulation (DR) in both ex vivo bone marrow-derived mesenchymal stromal cells (MSC) and in vitro 3T3-L1 preadipocytes significantly increases their adipogenic differentiation ability. Prep1 acts at a stage preceding the activation of the differentiation machinery because its DR makes cells more prone to adipogenic differentiation even in the absence of the adipogenic inducers. Prep1 DR expands the DNA binding landscape of C/EBPß (CCAAT enhancer binding protein ß) without affecting its expression or activation. The data indicate that Prep1 normally acts by restricting DNA binding of transcription factors to adipogenic enhancers, in particular C/EBPß.

Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression.

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

HOX and TALE signatures specify human stromal stem cell populations from different sources.

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues.