S861 LOSS OF THE F‐BOX PROTEIN NIPA CAUSES BONE MARROW FAILURE

Background: Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function and cancer susceptibility. It is a major diagnostic challenge to distinguish between IBMFS (e.g. Fanconi anemia (FA)), myelodysplastic syndrome (MDS) type refractory cytopenia of childhood (RCC) and severe aplastic anemia, due to overlapping phenotypes and pathological mechanisms, as well as, limited diagnostic tests and cells available. Aims: As therapeutic strategies differ in these entities, novel and clinical relevant biomarkers are urgently needed. Methods: To examine the functional role of NIPA in the hematopoietic stem cell (HSC) compartment in vivo , we analyzed Nipa −/− and Vav Tg/wt Nipa cko/cko mice. For human studies, we analyzed FA and RCC patients, which were enrolled in the European Working Group of MDS in Childhood (EWOG‐MDS) study. NIPA expression was determined by immunohistochemistry on trephine biopsy slides. Results: In our knockout mouse model, Nipa deletion shows both a reduction of the HSC pool and a cell intrinsic long‐term repopulation defect, with impaired self‐renewal in serial transplantations. Furthermore, Nipa −/− HSCs are unable to resolve DNA damage and prone to cell death. Interestingly, induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs. After short‐term Poly(I:C) treatment of young Nipa −/− mice we are able to show significantly reduced blood counts, diminished BM cellularity and lower LSK and LK levels. Even more remarkable are the results from the chronic stress assay: while control mice survive repeated Poly(I:C) injections for more than 400 days, all of the treated Vav Tg/wt Nipa cko/cko animals die due to complete BM failure, with a median latency of 354 days (p = 0.001). At the time of death, WBC counts are strongly reduced (p < 0.0001) and BM sections show substantial aplasia. Thus, loss of NIPA leads to a BM failure phenotype in mice resembling functional Fanc deficiency. Based on pathological MMC sensitivity in vitro and the FA‐like phenotype of Nipa −/− mice we postulated that Nipa deficiency might play a role in human disorders characterized by hypocellularity of the BM. We sequenced the NIPA gene in 5 patients with confirmed, but unclassified FA, who were negative for all so far described FA genes, but find no NIPA mutations in whole exome sequencing. We next focused on BM samples from children with MDS type RCC, a disease characterized by pancytopenia, BM dysplasia, normal blast percentage and often BM hypocellularity. As expected, FA patients with documented germline FANC gene mutation show physiological NIPA expression in their bone marrow. In contrast, only 13 out of 24 RCC patients show normal NIPA expression, while 11 patients display strongly reduced NIPA levels. Surprisingly, in all of the patients with defined germline mutation (i.e. in GATA2, SAMD9L or RUNX1) we find regular NIPA expression. Of the 18 patients without known genetic predisposition and with normal karyotype, 11 show a strong reduction in NIPA expression. Together with the findings in our mouse model this result indicates that a reduction of NIPA levels might contribute to hematopoietic failure in these patients. Summary/Conclusion: Our study identifies NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of pediatric RCC patients, thereby emphasizing its impact as potential diagnostic marker and/or possible therapeutic target.