Integrating Enhancer Profiling and CRISPR Dropout Screen Revealed Selenoprotein Synthesis Pathway As a New Vulnerability in AML

Abstract

Alterations of the epigenetic landscape and transcription are hallmarks of acute myeloid leukemia (AML) that drive leukemogenic gene expression and therefore can be exploited for therapeutic intervention. To look for such targets that harbor both an altered epigenetic feature and are genetically essential for AML cells, we performed a multi-database analysis integrating pan-cancer super enhancer landscapes with whole genome CRISPR dropout screens. Among the top targets, we discovered SEPHS2. An enhancer was present upstream of the gene marked by H3K27ac and bound by leukemogenic transcription factors including MYB, Pu.1 and RUNX1. In addition, AML cells with SEPHS2 deletion significantly dropped out in a genome wide CRISPR screen. This gene encodes a critical enzyme in the underappreciated selenoprotein synthesis pathway which was highly upregulated in TCGA AML patients compared to control blood cells from healthy individuals. Collectively, our initial bioinformatic analysis suggested that the selenoprotein synthesis pathway is a new vulnerability in AML. To test the functional requirement of the selenoprotein synthesis pathway in AML and other cells, we performed CRISPR mediated deletion of three key genes in the selenoprotein synthesis pathway, SEPHS2, SEPSECS and EEFSEC. The human AML cell lines (MOLM13, THP1 and Kasumi1), murine AML cells transformed by MLL-AF9 and human AML PDX cells all depicted a significant dependency on these genes, while proliferation of normal cord blood cells and myeloma cells (U266B1) was almost not affected. We then transplanted these cells into recipients. Deletion of SEPHS2, SEPSECS or EEFSEC significantly ameliorated AML progression, indicated by decreased AML burden and extended survival. Since selenoproteins including GPX1 and GPX4 are known antioxidants, we hypothesized that perturbing the selenoprotein synthesis pathway disrupted the redox state in AML cells. We found that deletion of SEPHS2, SEPSECS or EEFSEC elevated ROS in AML cells as demonstrated by Cell-Rox or DCF-DA staining. Western blotting revealed significantly downregulated GPX4 level and upregulated DNA damage marker γ-H2AX. Moreover, the defective proliferation was partially rescued by adding antioxidant TEMPOL. These results suggested selenoprotein synthesis pathway produced key antioxidants to balance the proper redox state and was required for AML cell proliferation. A major source of selenium is diet. Therefore, we hypothesized that consuming selenium low diet could suppress AML. We compared the survival of AML bearing mice on selenium proficient and deficient diet. The selenium deficient diet significantly extended survival, lowered GPX4 level and increased ROS in AML cells. Interestingly, normal mouse on selenium deficient diet for a 3-months period did not develop any abnormalities in CBC or bone marrow hematopoiesis. This suggests selenium deficient diet could be clinically applicable without significant side effects. Altogether, the integration of a pan-cancer enhancer landscape study with CRISPR dropout gene screen offered a powerful tool to dissect cancer targets that possessed unique enhancer features and genetic essentiality. The analysis yielded SEPHS2 and its selenoprotein synthesis pathway to be a new vulnerability in AML. The underappreciated selenoprotein synthesis pathway was key to produce antioxidant selenoproteins such as GPX1 and GPX4 to maintain a proper redox state in AML. Deleting the genes or removing selenium from diet could perturb the pathway and ameliorate the AML disease. Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

Therapeutic Benefits of Selenium in Hematological Malignancies

“…Mutational and expression-profiling studies can provide insights into the modified epigenetics or transcription pathways of AML patients to uncover key genes or pathways that could be targeted to inhibit leukemogenesis [ 168 ]. A bioinformatic analysis by Jing et.…”

“…A bioinformatic analysis by Jing et. al., involving pan-cancer super enhancer profiling and CRISPR dropout screens, revealed that the selenoprotein synthesis pathway may have critical implications towards AML [ 168 ]. The data from The Cancer Genome Atlas (TCGA) indicated that the SEPHS2 gene, which plays an important role in the selenoprotein synthesis pathway, is greatly upregulated in AML patients relative to healthy patients’ blood cells [ 168 ].…”

“…al., involving pan-cancer super enhancer profiling and CRISPR dropout screens, revealed that the selenoprotein synthesis pathway may have critical implications towards AML [ 168 ]. The data from The Cancer Genome Atlas (TCGA) indicated that the SEPHS2 gene, which plays an important role in the selenoprotein synthesis pathway, is greatly upregulated in AML patients relative to healthy patients’ blood cells [ 168 ]. The CRISPR deletion of genes critical to the synthesis of selenoproteins, such as SEPHS2 , SEPSECS , and EEFSEC in human AML cells (e.g., MOLM-13, Kasumi-1, THP-1, and patient-derived xenograft samples) and murine AML cells with an MLL-AF9 mutation exhibited a reduced proliferation and an enhanced production of ROS relative to healthy cord blood and myeloma cells (U266B1) [ 168 ].…”

“…The data from The Cancer Genome Atlas (TCGA) indicated that the SEPHS2 gene, which plays an important role in the selenoprotein synthesis pathway, is greatly upregulated in AML patients relative to healthy patients’ blood cells [ 168 ]. The CRISPR deletion of genes critical to the synthesis of selenoproteins, such as SEPHS2 , SEPSECS , and EEFSEC in human AML cells (e.g., MOLM-13, Kasumi-1, THP-1, and patient-derived xenograft samples) and murine AML cells with an MLL-AF9 mutation exhibited a reduced proliferation and an enhanced production of ROS relative to healthy cord blood and myeloma cells (U266B1) [ 168 ]. Transplantation studies of these modified AML cells into secondary recipients slowed the disease progression, reduced leukemia burden, and prolonged the survival [ 168 ].…”

“…A biochemical analysis of CRISPR-modified AML cells by Jiang and co-workers suggested that the deficiency to produce antioxidants, such as GPX1 and GPX4 , disrupted the redox environment necessary for the viability of AML cells, as evidenced by the reduced antioxidant Gpx4 and increased DNA damage marker γ -H2AX protein expression levels [ 168 ]. This was further supported by the slightly improved cellular proliferation of these CRISPR modified AML cells in the presence of the widely employed antioxidant and stable aminoxyl radical species, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) [ 168 ]. The AML murine models that received a diet deficient in selenium experienced a prolonged survival, reduced Gpx4 protein expression, and enhanced ROS production versus the control subjects that were fed a selenium adequate diet [ 168 ].…”