Genetic Modulators of Niclosamide Sensitivity and Resistance in Acute Myeloid Leukemia

Introduction

Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that leads to the accumulation of immature blasts in the blood and bone marrow. While the 5-year relative survival rate has increased from 6.4% to 28.7% in the last 40 years, death rates have remained steady at 3-4%. Less toxic, more effective targeted treatments are needed to improve outcomes for patients with AML. Previous studies identified cAMP response element-binding protein (CREB) as a potential therapeutic target for AML therapy. CREB is overexpressed in AML and is associated with poor prognosis. We identified a small molecule, XX-650-23, that inhibits interaction between CREB and its co-activator, CREB Binding Protein. However, XX-650-23 does not have optimal physical chemical properties for clinical application in humans. Niclosamide, an FDA approved anthelmintic drug, shares structural similarity with XX-650-23, and suppresses the proliferation of AML cells in vitro and in vivo by inhibiting CREB-dependent signaling. The aim of this study is to define the molecular pathways that mediate the effects of niclosamide in AML cells. This will allow for more precise selection of patient populations for treatment, identify potential sources of niclosamide resistance and reveal combination therapies that make use of the distinct pathways targeted.

Methods

To identify genetic factors modulating cellular sensitivity to niclosamide treatment, we performed a CRISPR/Cas9 library screen in the presence or absence of niclosamide. HL60 cells expressing Cas9 were infected with a custom sgRNA lentivirus library of 220 genes from a previous genome-wide shRNA screen. After puromycin selection, HL60 cells expressing pooled sgRNAs were grown in either niclosamide or 0.1% DMSO for 20 days. Genomic DNA was extracted from control and niclosamide-treated cells. Frequency of sgRNA were measured by next generation sequencing and analyzed using the Cas9 high-throughput maximum likelihood estimator (casTLE) algorithm.

Results

Sixty-five gene hits were found to be significant (p≤0.05). Of the top 220 hits from the previous genome-wide shRNA screen, 26 were identified as positive hits (p ≤0.05) in the CRISPR/Cas9 knock out screen. The CRISPR screen identified genes enriched in a number of pathways including programmed cell death, nucleotide biosynthesis, mitochondrial processes and canonical glycolysis, as measured by gene ontology term analysis. Numerous gene deletions conferred resistance to niclosamide treatment. Genes DHODH (score= 123; effect= 2.9) and HSPA9 (score= 144; effect = 2.5), involved in nucleotide biosynthesis and mitochondrial processes, were significantly enriched in the surviving population.

Perturbation of known metabolic pathways sensitized AML cells to niclosamide, including ATP synthase F1 subunit beta (ATP5B) (score= 316; effect= -5.1), hexokinase 2 (HK2) (score= 312; effect= -5.4), phosphofructokinase (PFKP) (score= 195; effect= -1.9) and mitochondrial phosphate carrier protein (SLC25A3) (score= 271; effect= -7.8). SLC25A3 and ATP5B play a role in oxidative phosphorylation and mitochondrial proton transmembrane transport, whereas HK2 and PFKP are key to glycolysis, suggesting that niclosamide's mechanism of action is dependent on energy metabolism. Furthermore, we found that disruption of Bcl-2 (BCL2) was sensitizing to niclosamide treatment (score= 129; effect= -4.7). To confirm the sensitizing effect of Bcl-2 inhibition on niclosamide treatment, HL60 cells were treated with niclosamide and Bc1-2 inhibitor venetoclax. Pretreatment of HL60 cells with niclosamide led to significantly decreased cell viability upon venetoclax treatment (p<0.0001). Simultaneous treatment of HL60 cells with niclosamide and venetoclax had a synergistic effect on cellular viability, with combination index values <1 at ED50 to ED90 (Chou-Talalay method).

Overall, our study identified genes with strong signatures for cell death, nucleotide biosynthesis, mitochondrial function and metabolic processes. Compounds inhibiting genes identified in this screen could be combined with niclosamide for synergistic effect. Targets overrepresented in the surviving population represent sources of resistance to niclosamide therapy. This study provides insight into mechanisms of action and resistance in AML cells treated with niclosamide and novel targets for combination therapy.