Characterizing the In Vivo impact of inflammation on Samd9l hematopoietic phenotypes. Free

Germline mutations in SAMD9 (sterile alpha motif domain containing 9) and SAMD9L (sterile alpha motif domain containing 9-like) are genetic predispositions to pediatric myeloid neoplasms, including 8-17% of children with MDS, and bone marrow failure (BMF) syndromes. These bone marrow abnormalities are often accompanied by deletions involving chromosome 7. SAMD9 and SAMD9L are located on chromosome 7, and the retained allele in bone marrow (BM) samples with monosomy 7 is universally wild-type, suggesting that there is selective pressure to not express mutant SAMD9/9L proteins. Using a Vav1-Cre conditional knock-in mouse model, we've shown that expression of mutant Samd9l (9l-Mut) results in a BMF phenotype, which is largely driven by increases in inflammatory pathways, such as TNFα and IFN signaling. This leads to cell death, which is worsened by exposure to inflammation via treatment with polyinosinic:polycytidylic acid (pIpC). Samd9l is located on chromosome 6 in mice, and we've also observed chromosome 6 deletions (Mo6) in mice treated with pIpC, recapitulating the observations in patients. Despite these findings, the role of inflammatory pathways on Samd9l-Mut-dependent phenotypes remains unclear. Furthermore, the mechanisms by which genomic alterations, such as Mo6, promote survival in 9l-Mut mice after inflammatory insults are poorly understood. We hypothesize that inflammation is a targetable pathway that is required for 9l-Mut-dependent BMF, chromosomal 6 deletions, and transcriptional repression of Samd9l mutant-dysregulated pathways.

To assess the role of inflammation on phenotypes, 9l-Mut mice (n=62) and controls (n=33) were treated with pIpC or vehicle for 4 weeks and aged for 300 days. BM was isolated for hematopoietic and transcriptional characterization. To further assess the role of inflammation on the development of BMF, we crossed our 9l-Mut strain with (B6(Cg)-Ifnar1tm1.2Ees/J). We treated this new strain (9l-Mut/Ifnar1-KO) with and without pIpC for 4 weeks, and BM was isolated for hematopoietic and transcriptional characterization.

While ~30% of vehicle-treated 9l-Mut mice die from BMF, pIpC treated mice have a median survival of 92.5 days (p=<0.0001). 9l-Mut mice die with hypocellular BM with marked depletion of LT-HSCs. Interestingly, aged 9l-Mut mice that survive until day 300 have normal cellularity with myeloid skewing. We performed WGS and RNA-seq on HSPCs to assess chromosomal deletions and transcriptional changes that promote long-term survival. 1 of 4 surviving mice used for WGS had a heterozygous deletion of chromosome 6 with no observed LOH or cis mutations in Samd9l. We also saw no significant changes in the transcriptional profile between aged Samd9l-WT and aged 9l-Mut mice, while BMF 9l-Mut mice show significant changes in translation, cell cycle, and DNA damage pathways. To assess the hematopoietic capacity of HSPCs with Mo6, we transplanted WBM cells from Mo6 mice and controls into lethally irradiated mice. HSPCs from Mo6 mice were able to partially repopulate the BM, while 9l-Mut HSPCs were significantly less fit and failed repopulation.

To assess the genetic rescue of inflammation, we treated 9l-Mut/Ifnar1-KO and control mice with and without pIpC for 4 weeks, then assessed the hematopoietic compartment. The BM cellularity of treated 9l-Mut/Ifnar1-KO mice was rescued when compared to treated 9l-Mut mice, and LT-HSCs; however, when compared to WT mice, 9l-Mut/Ifnar1-KO mice still have a myeloid bias.

Our data highlights the importance of inflammation in Samd9l BM phenotypes. Inflammation causes a significantly shortened BMF latency while simultaneously leading to monosomy 6, removing the mutant allele, which provides a fitness advantage. Genetic rescue of inflammation suggests that targeting inflammatory pathways could be critical for the survival of patients with SAMD9/SAMD9L syndromes. While we show that inflammation drives 9l-Mut-dependent chromosomal deletions, which promote survival, it is still unclear what drives transcriptional repression of inflammatory pathways to promote survival in mutant mice, and how deletions impact disease progression. Further characterization of these novel models will be critical in understanding disease progression as well as therapeutic approaches.