αB-Crystallin Peptide Fused with Elastin-like Polypeptide: Intracellular Activity in Retinal Pigment Epithelial Cells Challenged with Oxidative Stress.

BACKGROUND: Oxidative stress-induced retinal degeneration is among the main contributing factors of serious ocular pathologies that can lead to irreversible blindness. αB-crystallin (cry) is an abundant component of the visual pathway in the vitreous humor, which modulates protein and cellular homeostasis. Within this protein exists a 20 amino acid fragment (mini-cry) with both chaperone and antiapoptotic activity. This study fuses this mini-cry peptide to two temperature-sensitive elastin-like polypeptides (ELP) with the goal of prolonging its activity in the retina. METHODS: The biophysical properties and chaperone activity of cry-ELPs were confirmed by mass spectrometry, cloud-point determination, and dynamic light scattering 'DLS'. For the first time, this work compares a simpler ELP architecture, cry-V96, with a previously reported ELP diblock copolymer, cry-SI. Their relative mechanisms of cellular uptake and antiapoptotic potential were tested using retinal pigment epithelial cells (ARPE-19). Oxidative stress was induced with H2O2 and comparative internalization of both cry-ELPs was made using 2D and 3D culture models. We also explored the role of lysosomal membrane permeabilization by confocal microscopy. RESULTS: The results indicated successful ELP fusion, cellular association with both 2D and 3D cultures, which were enhanced by oxidative stress. Both constructs suppressed apoptotic signaling (cleaved caspase-3); however, cry-V96 exhibited greater lysosomal escape. CONCLUSIONS: ELP architecture is a critical factor to optimize delivery of therapeutic peptides, such as the anti-apoptotic mini-cry peptide; furthermore, the protection of mini-cry via ELPs is enhanced by lysosomal membrane permeabilization.

Deletion of CD36 exhibits limited impact on normal hematopoiesis and the leukemia microenvironment.

BACKGROUND: CD36 has been identified as a potential therapeutic target both in leukemic cells and in the tumor immune microenvironment. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. To establish CD36 as a viable therapeutic target in AML, we investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. METHODS: Differential expression data of CD36 during human and mouse normal hematopoiesis were examined and compared. Cd36 knockout (Cd36-KO) mice were evaluated for blood analysis, hematopoietic stem cells and progenitors (HSPCs) function and phenotype analyses, and T cells in vitro expansion and phenotypes in comparison with wild type (WT) mice. In addition, MLL-PTD/FLT3-ITD leukemic cells were engrafted into Cd36-KO and WT mice, and leukemia burden was compared between groups. RESULTS: RNA-Seq data showed that Cd36 expression was low in HSPCs and increased as cells matured. Phenotypic analysis revealed limited changes in blood count except for a slight yet significantly lower red blood cell count and hemoglobin and hematocrit levels in Cd36-KO mice compared with WT mice (P < 0.05). In vitro cell proliferation assays of splenocytes and HSPCs from Cd36-KO mice showed a similar pattern of expansion to that of cells from WT mice. Characterization of HSPCs showed similar percentages of the different progenitor cell populations between Cd36-KO with WT mice. However, Cd36-KO mice exhibited ~ 40% reduction of the number of colonies developed from HSPCs cells compared with WT mice (P < 0.001). Cd36-KO and WT mice presented comparably healthy BM transplant in non-competitive models and developed similar leukemia burden. CONCLUSIONS: Although the loss of Cd36 affects the hematopoietic stem cell and erythropoiesis, limited detrimental overall impact was observed on normal Hematopoietic and leukemic microenvironments. Altogether, considering the limited impact on normal hematopoiesis, therapeutic approaches to target CD36 in cancer are unlikely to result in toxicity to normal blood cells.

Strategies targeting FLT3 beyond the kinase inhibitors.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion and differentiation arrest of the myeloid progenitor cells, which leads to the accumulation of immature cells called blasts in the bone marrow and peripheral blood. Mutations in the receptor tyrosine kinase FLT3 occur in 30% of normal karyotype patients with AML and are associated with a higher incidence of relapse and worse survival. Targeted therapies against FLT3 mutations using small-molecule FLT3 tyrosine kinase inhibitors (TKIs) have long been investigated, with some showing favorable clinical outcomes. However, major setbacks such as limited clinical efficacy and the high risk of acquired resistance remain unresolved. FLT3 signaling, mutations, and FLT3 inhibitors are topics that have been extensively reviewed in recent years. Strategies to target FLT3 beyond the small molecule kinase inhibitors are expanding, nevertheless they are not receiving enough attention. These modalities include antibody-based FLT3 targeted therapies, immune cells mediated targeting strategies, and approaches targeting downstream signaling pathways and FLT3 translation. Here, we review the most recent advances and the challenges associated with the development of therapeutic modalities targeting FLT3 beyond the kinase inhibitors.