Isoform-specific, Semi-quantitative Determination of Highly Homologous Protein Levels via CRISPR-Cas9-mediated HiBiT Tagging.

Many protein families consist of multiple highly homologous proteins, whether they are encoded by different genes or originating from the same genomic location. Predominance of certain isoforms has been linked to various pathological conditions, such as cancer. Detection and relative quantification of protein isoforms in research are commonly done via immunoblotting, immunohistochemistry, or immunofluorescence, where antibodies against an isoform-specific epitope of particular family members are used. However, isoform-specific antibodies are not always available, making it impossible to decipher isoform-specific protein expression patterns. Here, we describe the insertion of the versatile 11 amino acid HiBiT tag into the genomic location of the protein of interest. This tag was developed and is distributed by Promega (Fitchburg, WI, USA). This protocol describes precise and specific protein expression analysis of highly homologous proteins through expression of the HiBiT tag, enabling protein expression quantification when specific antibodies are missing. Protein expression can be analyzed through traditional methods such as western blotting or immunofluorescence, and also in a luciferase binary reporter system, allowing for reliable and fast relative expression quantification using a plate reader. Graphical overview

Lysosomes in acute myeloid leukemia: potential therapeutic targets?

Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules. However, in recent studies, they have begun to emerge as crucial regulators of cell homeostasis. They are at the crossroads of catabolic and anabolic pathways and are intricately involved in cellular trafficking, nutrient signaling, energy metabolism, and immune regulation. Their involvement in such essential cellular functions has renewed clinical interest in targeting the lysosome as a novel way to treat disease, particularly cancer. Acute myeloid leukemia (AML) is an aggressive blood cancer with a low survival probability, particularly in older patients. The genomic landscape of AML has been extensively characterized but few targeted therapies (with the exception of differentiation therapy) can achieve a long-term cure. Therefore, there is an unmet need for less intensive and more tolerable therapeutic interventions. In this review, we will give an overview on the myriad of functions performed by lysosomes and their importance in malignant disease. Furthermore, we will discuss their relevance in hematopoietic cells and different ways to potentially target them in AML

HSPA8 Chaperone Complex Drives Chaperone-Mediated Autophagy Regulation in Acute Promyelocytic Leukemia Cell Differentiation.

INTRODUCTION Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. METHODS We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. RESULTS Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. CONCLUSION Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones