Identification of genetic targets in acute myeloid leukaemia for designing targeted therapy

Abstract

Summary Few effective therapies exist for acute myeloid leukaemia (AML), in part due to the molecular heterogeneity of this disease. We sought to identify genes crucial to deregulated AML signal transduction pathways which, if inhibited, could effectively eradicate leukaemia stem cells. Due to difficulties in screening primary cells, most previous studies have performed next‐generation sequencing (NGS) library knockdown screens in cell lines. Using carefully considered methods including evaluation at multiple timepoints to ensure equitable gene knockdown, we employed a large NGS short hairpin RNA (shRNA) knockdown screen of nearly 5 000 genes in primary AML cells from six patients to identify genes that are crucial for leukaemic survival. Across various levels of stringency, genome‐wide bioinformatic analysis identified a gene in the NOX family, NOX1, to have the most consistent knockdown effectiveness in primary cells (P = 5∙39 × 10−5, Bonferroni‐adjusted), impacting leukaemia cell survival as the top‐ranked gene for two of the six AML patients and also showing high effectiveness in three of the other four patients. Further investigation of this pathway highlighted NOX2 as the member of the NOX family with clear knockdown efficacy. We conclude that genes in the NOX family are enticing candidates for therapeutic development in AML.

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis

“…In the HEL cell line model, the essential role of the proteasomal gene family for viability was observed regardless of treatment with ruxolitinib or CM exposure. Our previous pooled-shRNA library screen of primary AML cells using the identical library did not show proteasome genes among targets having greatest depletion [ 21 ], indicating that proteasome genes are not the most effective targets across all myeloid malignancies. Furthermore, chemical validation demonstrated that relatively low doses of proteasome inhibitors were able to significantly affect HEL cell viability, validating the pooled-shRNA library screen results.…”

“…Samples were randomized prior to sequencing on a NextSeq 500 Sequencing System (Illumina, San Diego, CA, USA) to reduce potential biases associated with variable sequencing depths and other NGS-related artefacts [ 20 ]. Bioinformatic analyses and fold change calculations were performed as previously described [ 17 , 21 ]. In brief, the barcodes for each shRNA were identified in the sequencing data for each sample.…”

Genomic Abnormalities as Biomarkers and Therapeutic Targets in Acute Myeloid Leukemia

“…For instance, the Beat AML clinical trial was initiated based on the concept of using genomic technology to identify each patient's cancerdriving genetic mutations, followed by matching patients with the most promising targeted treatment, and the initial observations have been promising [11]. Alternatively, one can use inhibition using small hairpin RNA (shRNA) or CRISPR technology libraries [159][160][161][162]. For the last few years, pooled shRNA libraries have been applied to various cancer cell lines in combination with various drugs to identify the genes that are essential for the viability of the cancer cells, and those whose inhibition demonstrates synthetic lethality with the investigated inhibitor [160,163].…”

“…During the selection period, the shRNAs targeting the genes essential for survival of the investigated cancer cells are depleted. The depleted shRNAs are then characterised using next generation sequencing techniques [160,161,163].…”

“…The main challenges on screening primary cells are the low efficiency of the current techniques at transducing primary cells with shRNA or CRISPR vectors, and also the short in vitro survival of the primary cells, which is particularly true for AML [164]. We were able to screen primary AML cells in two independent investigations using a selective pooled shRNA library (selected from reviewing literature on the genes involved in AML pathogenesis and also from high throughput sequencing data) [165], and then a pooled library targeting genes known to be involved in major signalling pathways [161], which identified a few essential genes for the viability in some AML cases. However, further modification to the methodology and larger numbers of AML cases with various subtypes are required before validation of this method as a clinically approved tool for therapeutic target discovery in patients.…”