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Chemotherapy Resistance Pathway for AML Identified in CRISPRa Screen

NEW YORK (GenomeWeb) – An international team of researchers has used a CRISPR activation (CRISPRa) screen to identify both coding and noncoding pathways involved in acquired resistance to cytarabine, the main treatment for acute myeloid leukemia (AML).

The study, led by senior author Pier Paolo Pandolfi of the Beth Israel Deaconess Medical Center and Harvard Medical School, was published in Cell today.

About 30 percent to 50 percent of AML patients relapse with chemotherapy-resistant disease. In order to determine the mechanisms behind this, the researchers began by identifying putative resistance genes using pharmacogenetic data from 760 human pan-cancer cell lines from the Cancer Cell Line Encyclopedia. After that, they functionally characterized both coding and long non-coding RNA (lncRNA) genes on a genome scale using CRISPRa screens. To assess the function of lncRNAs, they developed a strategy called CRISPR activation of lncRNA (CaLR) in which they targeted 14,701 lncRNA genes and performed computational and functional analyses to identify novel cell-cycle, survival or apoptosis, and cancer signaling genes.

"Transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML," the authors wrote. "Thus, [dual protein-coding and non-coding integrated CRISPRa screening] DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance."

To define biological pathways predictive of resistance to cytarabine, the team performed a gene-set enrichment analysis and identified positive enrichment of cell survival signaling pathways, including the Jak-STAT, PI3K-Akt, and MAPK pathways, and negative enrichment of the pyrimidine metabolic pathway, mechanisms related to DNA damage, and RNA regulatory mechanisms.

In order to functionally validate that predictive analysis in a high-throughput manner, the researchers established a CRISPRa-based system in AML cell lines to analyze the coding and non-coding genes contributing to cytarabine resistance. They treated cells for 14 days with the chemotherapeutic agent and then analyzed the abundances of single-guide RNAs in the cells to identify which genes had been depleted or enriched.

Both the correlation analysis and the forward genetic screen found DCK to be the most significantly depleted gene, indicating that strong transcriptional activation of DCK by CRISPRa confers high sensitivity to cytarabine, the researchers said. They also identified multiple genes suspected to modulate sensitivity to cytarabine. "Importantly, we identified a large overlap of 2,411 genes significantly enriched/depleted in both our cell line and protein-coding CRISPRa screening," they wrote. "We subsequently validated a subset of these genes, including ZBP1, MUL1, and PI4K2A, whose expression was associated with poor prognosis and decreased disease-free survival."

The team went in to study the functional roles of lncRNA genes in cytarabine resistance by designing an sgRNA library of 14,701 lncRNA genes, resulting in a library of 88,444 targeting guides. The resulting screening experiments led to a short list of novel annotated lncRNAs which were significantly enriched in both the functional screening and the cell line analysis. Subsequent co-expression analysis of the individual lncRNA transcript levels with their most highly correlated protein coding genes suggested to the researchers that these lncRNAs play roles in survival pathways know to affect leukemia and drug resistance.

In order to validate the findings, they chose 11 genes that were significantly enriched and two genes that were significantly depleted in their screens for further characterization. They treated cells expressing the relevant sgRNAs with cytarabine and found that expression of each enriched sgRNA resulted in decreased cytarabine sensitivity. The depleted lncRNA genes also behaved as expected, the team said. Further analysis pointed to GAS6-AS2 as having a significant ability to attenuate apoptosis.

In a subsequent computational analysis, the team found that GAS6-AS2 functions through cis-regulation of its adjacent cognate gene, coding for the GAS6 ligand, as well as the trans-regulation of its receptor, AXL, to drive aberrant downstream signaling of this pathway.

"While each of these approaches as individual modules (computational and screening) has been shown to be useful to identify genes regulating specific cellular processes, each harbors inherent limitations and bias requiring extensive validation of hits," the authors concluded. "However, our integrated approach described here serves as a more powerful framework for the screening and discovery of protein-coding and non-coding networks regulating biological processes, thereby providing a resource to facilitate improvements toward the annotation and functionalization of non-coding RNAs at large."