Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes

Neggers, Jasper E.; Kwanten, Bert; Dierckx, Tim; Noguchi, Hideyuki; Voet, Arnout; Bral, Lotte; Minner, Kristien; Massant, Bob; Kint, Nicolas; Delforge, Michel; Vercruysse, Thomas; Baloglu, Erkan; Senapedis, William; Jacquemyn, Maarten; Daelemans, Dirk

doi:10.1038/s41467-017-02349-8

Cited by 91 publications

(74 citation statements)

References 46 publications

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“…Corroborating our findings, NAMPT was recently identified as the main target of KPT-9274 by using a CRISPR-Cas9 mutagenesis scan. 50 This previous publication and our work described herein firmly establish NAMPT as a KPT-9274-relevant target in AML.…”

Section: Discussionsupporting

confidence: 69%

Selective targeting of NAMPT by KPT-9274 in acute myeloid leukemia

Mitchell¹,

Larkin²,

Grieselhuber³

et al. 2019

Blood Advances

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Key Points• KPT-9274, via its protein target NAMPT, diminishes NAD 1 levels and cellular respiration, leading to cell death.• Orally bioavailable KPT-9274 exhibits targetspecific activity in cell lines and patientderived xenograft models of AML. Treatment options for acute myeloid leukemia (AML) remain extremely limited and associated with significant toxicity. Nicotinamide phosphoribosyltransferase (NAMPT) is involved in the generation of NAD 1 and a potential therapeutic target in AML. We evaluated the effect of KPT-9274, a p21-activated kinase 4/NAMPT inhibitor that possesses a unique NAMPT-binding profile based on in silico modeling compared with earlier compounds pursued against this target. KPT-9274 elicited loss of mitochondrial respiration and glycolysis and induced apoptosis in AML subtypes independent of mutations and genomic abnormalities. These actions occurred mainly through the depletion of NAD 1 , whereas genetic knockdown of p21-activated kinase 4 did not induce cytotoxicity in AML cell lines or influence the cytotoxic effect of KPT-9274. KPT-9274 exposure reduced colony formation, increased blast differentiation, and diminished the frequency of leukemia-initiating cells from primary AML samples; KPT-9274 was minimally cytotoxic toward normal hematopoietic or immune cells. In addition, KPT-9274 improved overall survival in vivo in 2 different mouse models of AML and reduced tumor development in a patient-derived xenograft model of AML.Overall, KPT-9274 exhibited broad preclinical activity across a variety of AML subtypes and warrants further investigation as a potential therapeutic agent for AML.

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Section: Discussionsupporting

confidence: 69%

Selective targeting of NAMPT by KPT-9274 in acute myeloid leukemia

Mitchell¹,

Larkin²,

Grieselhuber³

et al. 2019

Blood Advances

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“…Essential protein domains are often associated with hyper-sensitivity to CRISPR/Cas9 knockouts, permitting in situ analysis of protein functions. Previous applications have mainly focused on the validation of annotated or hypothetical domains 5,7 . A pooled CRISPR screen allows a tiling-sgRNA design for more than 100 proteins, holding promise for de novo discovery of essential domains.…”

Section: Discussionmentioning

confidence: 99%

“…Munoz et al performed the first high-throughput tilling-sgRNA screen on 159 genes, and confirmed that the sgRNAs that target pharmaceutically important protein domains are associated with stronger knockout effects 6 . Recently, a method combining tiling-sgRNA screens with positive selections has been developed to identify small-molecule drug target sites 7 . A computational pipeline, CRISPRO, maps functional scores of tiling sgRNAs to genomes, transcripts, protein coordinates and structures, providing general views of structure-function relationships at discrete protein regions 8 .…”

Section: Introductionmentioning

confidence: 99%

De novo Identification of Essential Protein Domains from CRISPR/Cas9 Tiling-sgRNA Knockout Screens

Zhang

Villarreal

et al. 2019

Preprint

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High-throughput CRISPR/Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. To facilitate de novo identification of essential protein domains from such screens, we developed ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refers to the protein regions that are associated with strong sgRNA dropout effect in the screens. We used ProTiler to analyze a published CRISPR tiling screen dataset, and identified 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlapped with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also revealed unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which we validated experimentally. Surprisingly, the CKHS regions were negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR/Cas9 mediated amino acids loss.

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“…The PAK4-NAMPT dual inhibitors (KPT-9274 and analog KPT-7523) were obtained from Karyopharm Therapeutics (Newton, MA, USA). KPT-9274 is a CRISPRres validated NAMPT inhibitor with dual inhibitory activity of PAK4 [40]. The following drugs were purchased from Selleckchem (Houston, TX, USA): Everolimus ((RAD001) mTOR inhibitor), INK-128, FK866 (APO866/Daporinad specific inhibitor of NAMPT), and PF3758309 (specific inhibitor of PAK4).…”

Section: Cell Lines and Reagentsmentioning

confidence: 99%

PAK4-NAMPT Dual Inhibition as a Novel Strategy for Therapy Resistant Pancreatic Neuroendocrine Tumors

Mpilla

Aboukameel

Muqbil

et al. 2019

Cancers

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Pancreatic neuroendocrine tumors (PNET) remain an unmet clinical need. In this study, we show that targeting both nicotinamide phosphoribosyltransferase (NAMPT) and p21-activated kinase 4 (PAK4) could become a synthetic lethal strategy for PNET. The expression of PAK4 and NAMPT was found to be higher in PNET tissue compared to normal cells. PAK4-NAMPT dual RNAi suppressed proliferation of PNET cell lines. Treatment with KPT-9274 (currently in a Phase I trial or analogs, PF3758309 (the PAK4 selective inhibitor) or FK866 (the NAMPT inhibitor)) suppressed the growth of PNET cell lines and synergized with the mammalian target of rapamycin (mTOR) inhibitors everolimus and INK-128. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers. KPT-9274 suppressed NAD pool and ATP levels in PNET cell lines. Metabolomic profiling showed a statistically significant alteration in cellular energetic pathways. KPT-9274 given orally at 150 mg/kg 5 days/week for 4 weeks dramatically reduced PNET sub-cutaneous tumor growth. Residual tumor analysis demonstrated target engagement in vivo and recapitulated in vitro results. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNET that warrant further clinical investigation.

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Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes

Cited by 91 publications

References 46 publications

Selective targeting of NAMPT by KPT-9274 in acute myeloid leukemia

Selective targeting of NAMPT by KPT-9274 in acute myeloid leukemia

De novo Identification of Essential Protein Domains from CRISPR/Cas9 Tiling-sgRNA Knockout Screens

PAK4-NAMPT Dual Inhibition as a Novel Strategy for Therapy Resistant Pancreatic Neuroendocrine Tumors

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