0142: A Systemic Autoinflammatory Syndrome Caused by NEMO Exon 5 Skipping

Bin Lin¹, Adriana Almeida de Jesus², Eric Karlins¹, Dana Kahle¹, Sophia Park³, Andre Rastegar¹, Jacob Mitchell⁴, sofia Torregiani¹, Farzana Bhuyan⁵, Sara Alehashemi⁶, Kader Cetin Gedik⁷, Kat Uss⁸, Eric Hanson⁹, Amer Khojah¹⁰, Eveline Wu¹¹, Christiaan Scott¹², Timothy Ronan Leahy¹³, Emma J. MacDermott¹³, Orla G. Killeen¹³, Chyi-Chia Richard Lee¹⁴, Thaschawee Arkachaisri¹⁵, Zoran Gucev¹⁶, Kathryn Cook¹⁷, Vafa Mammadova¹⁸, Gulnara Nasrullayeva¹⁸, Mariana Correia Marques¹⁹, Abigail Bosk²⁰, Seza Ozen²¹, Abigail Lang²², Brian Nolan²³, Scott Canna²⁴, Maude Tusseau²⁵, Emilie Chopin²⁵, Guilaine Boursier²⁶, Danielle Fink²⁷, Douglas Kuhns²⁷, Clifton Dalgard²⁸, Alexandre Belot²⁹, Timothy Moran³⁰, Andrew Oler³ and Raphaela Goldbach-Mansky³¹, ¹NIH, Bethesda, MD, ²NIAID, NIH, Silver Spring, MD, ³NIAID, Bethesda, MD, ⁴NIAID NIH, Bethesda, MD, ⁵National Institutes of Health, Bethesda, MD, ⁶NIH/NIAID/TADS, Clarksville, MD, ⁷Translational Autoinflammatory Diseases Section (TADS)/NIAID/NIH, Bethesda, MD, ⁸National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, ⁹Indiana University School of Medicine, Indianapolis, IN, ¹⁰Umm Al-Qura University, Makkah, Saudi Arabia, ¹¹University of North Carolina, Chapel Hill, NC, ¹²University of Cape Town, Red Cross War Memorial Children's Hospital, Cape Town, South Africa, ¹³CHI at Crumlin, Dublin, Ireland, ¹⁴National Cancer Institute, National Institutes of Health, Bethesda, MD, ¹⁵KK Women's and Children's Hospital, SingHealth, Singapore, Singapore, ¹⁶University Children's Hospital, Medical Faculty Skopje, Skopje, Macedonia, ¹⁷Akron Children's Hospital, Akron, OH, ¹⁸Azerbaijan Medical University, Baku, Azerbaijan, ¹⁹National Institute of Arthritis and Musculoskeletal and Skin Diseases / Children`s National Hospital, Bethesda, MD, ²⁰Children's National Hospital, Washington, DC, ²¹Hacettepe University Faculty of Medicine, Ankara, Turkey, ²²Lurie Children's Hospital, Chicago, IL, ²³Ann & Robert H. Lurie Children's Hospital, Chicago, IL, ²⁴Children's Hospital of Philadelphia, Philadelphia, PA, ²⁵Service de Néphrologie, Rhumatologie, Dermatologie Pédiatriques, Centre de Référence des Rhumatismes Inflammatoires et Maladies Auto-Immunes Rares de l'Enfant (RAISE), Hôpital Femme-Mère-Enfant, Hospices Civils de Lyon, Bron, France, ²⁶Laboratoire de Génétique des Maladies Rares et Autoinflammatoires, Département de Génétique Médicale, Maladies Rares et Médecine Personnalisée, CEREMAIA, CHU de Montpellier, Univ Montpellier, Montpellier, France, ²⁷National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD, ²⁸The American Genome Center, Uniformed Services University of the Health Sciences, Bethesda, MD, ²⁹Hospices Civils de Lyon, Collonges au mont d'or, France, ³⁰University of North Carolina School of Medicine, Chapel Hill, NC, ³¹NIH/NIAID, Potomac, MD

Background/Purpose: NF-kB essential modulator (NEMO, encoded by IKBKG) is an essential gene in immune response, development, and cell death regulation. Mutations in NEMO have been associated with 3 Mendelian phenotypes with developmental defects and/or immunodeficiencies but a systemic inflammation phenotype was rarely reported. Here we identified patients with systemic inflammation caused by splice variants resulting in exon 5 skipping of NEMO. We hypothesized that TNF induced cells death may be enhanced in these patients with the goal to identify therapeutic options and explore the role of cell death in causing inflammation.

Methods: NEMO exon 5-intro 5 boundaries were edited in U937 cells to mimic the NEMO exon 5 skipping in patients (pts). TNF induced cell death was measured by CellTiter-Glo.

Results: We identified a group of 18 pts (14 females and 4 males) with 10 different splice site variants causing NEMO exon 5 skipping. Common clinical features include panniculitis with systemic inflammation (100%), ectodermal dysplasia (83%), hepatosplenomegaly (77%) and B-cell lymphopenia (80%). We named this disease as NEMO deleted exon 5 autoinflammatory syndrome (NEMO-NDAS). Cell death was observed in skin and liver biopsies. Moreover, enhanced levels of soluble TNFR1 and TNFR2 were detected in serum compared to healthy controls, which further supported a role of cell death in the pathology. To understand the disease mechanism, U937 cell line with NEMO exon 5 skipping was created by CRISPR editing. This mutant cell line is highly susceptible to TNF induced cell death compared to the wildtype U937 cells. The cell death can be rescued by the combination of RIPK1 inhibitor Nec1s and CASP8 inhibitor Z-IETD-FMK, which indicates a RIPK1- and CASP8-dependent mechanism. NEMO is the central hub regulating TNF-induced TBK1 activation, IKKA/B activation and NFKB activation, which are important cell death checkpoints in the TNF pathway. Applying inhibitor for one of the checkpoints can further enhance TNF-induced cell death in mutant U937 cells, which suggests NEMO-NDAS mutation only led to partial loss-of-function in the three cell death checkpoints. Combination of the three inhibitors, however, led to increased cell death in wildtype U937 cells, which is comparable to TNF-stimulated mutant cells without inhibitors and indicates that NEMO-NDAS mutations cause partial loss-of-function in NEMO-mediated TBK1 activation, IKKA/B activation and NFKB target gene expression, which is mimicked by combining the 3 respective checkpoint inhibitors. The cell death in mutant cells can be rescued by TNF inhibitor Adalimumab or anti-TNFR1 antibody in a dose-dependent way and can be further enhanced by co-administration of the RIPK1 inhibitor Nec1s.

Conclusion: Our study showed that NEMO exon 5 deletion mutations in NEMO-NDAS patients lead to susceptibility to TNF induced cell death that is RIPK1- and CASP8-dependent and can be rescued by RIPK1 inhibitor, TNF inhibitor, and TNFR1 inhibitor, which provide novel therapeutic options for treating these patients.

Disclosures: B. Lin, None; A. Almeida de Jesus, None; E. Karlins, None; D. Kahle, None; S. Park, None; A. Rastegar, None; J. Mitchell, None; s. Torregiani, None; F. Bhuyan, None; S. Alehashemi, None; K. Cetin Gedik, None; K. Uss, None; E. Hanson, None; A. Khojah, None; E. Wu, Pharming Healthcare Inc, AstraZeneca, Bristol-Myers Squibb(BMS), Janssen; C. Scott, None; T. Leahy, None; E. MacDermott, None; O. Killeen, None; C. Lee, None; T. Arkachaisri, None; Z. Gucev, None; K. Cook, None; V. Mammadova, None; G. Nasrullayeva, None; M. Correia Marques, None; A. Bosk, None; S. Ozen, None; A. Lang, None; B. Nolan, Sobi; S. Canna, Sobi, Ab2bio, Novartis, Immvention Therapeutics, sobi, Simcha Therapeutics; M. Tusseau, None; E. Chopin, None; G. Boursier, None; D. Fink, None; D. Kuhns, None; C. Dalgard, None; A. Belot, SOBI, Novartis, Roche, Pfizer; T. Moran, None; A. Oler, None; R. Goldbach-Mansky, None.