Genome-wide CRISPR Screens Decipher Previously Unrecognized Modulators to Boost CAR-NK Cell Antitumour Potency — ASN Events
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Genome-wide CRISPR Screens Decipher Previously Unrecognized Modulators to Boost CAR-NK Cell Antitumour Potency (#168)

Alexander Biederstädt 1 2 , Rafet Basar 1 , Jeong-Min Park 1 , John G Watts 1 , Nadima Uprety 1 , Natalie Wall Fowlkes 3 , Rejeena Shrestha 1 , Francia Reyes Silva 1 , May Daher 1 , Hind Rafei 1 , Patrick Zhang 1 , Donghai Xiong 1 , Huihui Fan 4 , Paul Lin 1 , Enli Liu 1 , Ken Chen 5 , Bin Liu 1 , Pinaki Prosad Banerjee 1 , Silvia Tiberti 1 , Ping Li 1 , Sanjida Tanver Islam 1 , Vernikka C Woods 1 , Inci Biederstädt 1 , Xin Ru Jiang 1 , Sunil Acharya 1 , Merve Dede 5 , Seema Rawal 1 , Ana Karen Nunez Cortes 1 , Ye Li 1 , Luis H Muniz Feliciano 1 , Katy Rezvani 1 , Elizabeth Shpall 1
  1. Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
  2. Department of Medicine III - Hematology & Medical Oncology, Technical University of Munich, Munich, BY, Germany
  3. Department of Veterinary Medicine & Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
  4. Department of Neurology, The University of Texas McGovern Medical School, Houston, TX, United States
  5. Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States

Natural killer (NK) cells have emerged as a promising new treatment avenue. However, intrinsic and extrinsic barriers may impinge on their therapeutic efficacy, and strategies to overcome these challenges remain largely unexplored. To address this, we developed a pooled CRISPR discovery platform to identify genetic perturbations that can be harnessed for CAR-NK cell engineering.

 

We conducted multiple genome-wide CRISPR screens in primary human NK cells under immunosuppressive conditions and repeated tumour challenge. These screens uncovered novel NK cell regulators which modulate resistance to tumour-induced and TME-driven immunosuppression. High-content screening in NK cells with enhanced degranulation capacity validated these findings, identifying phenotypic correlates of enhanced potency. Cross-validation in a large patient cohort across 17 diverse cancer types revealed transcriptional upregulation of top screen hits compared to healthy donors, highlighting their clinical relevance.

 

Key pathways enriched among the hits included components of the Cul5-RING ubiquitin ligase, the Mediator and the SAGA complexes, as well as genes orchestrating chromatin remodelling and regulation of NFκB. These findings highlight transcriptional reprogramming and post-translational modification (PTM) as key modulators of NK cell effector function.

 

Disruption of MED12, ARIH2, and CCNC profoundly improved the antitumour potency of both non-transduced and CAR-transduced NK cells against multiple hard-to-treat cancers using two IL-15-armoured CARs targeting CD70 and TROP2. ARIH2- and CCNC-deficient TROP2-CAR-NK cells displayed enhanced metabolic fitness, accompanied by induction of cytotoxic subsets, as revealed by mass cytometry.

 

Multiplexed ARIH2/CCNC editing in TROP2-CAR-NK cells significantly boosted NK cell expansion, tumour bed infiltration, cytotoxic granule release, and antitumour efficacy in an orthotopic pancreatic cancer mouse model.

 

In conclusion, high-content genome-wide CRISPR screening in primary human NK cells identified novel regulators of NK cell function, including MED12, ARIH2, and CCNC. Targeting these checkpoints enhances CAR-NK cell metabolic fitness, cytotoxicity, and in vivo antitumour efficacy, paving the way for next-generation CAR-NK immunotherapies.