Citations

Abstract Germline variants in DDX41 are the most frequent genetic predisposition to adult hematologic malignancies. The most common variants are truncating, implicating loss of function in the pathogenesis. However, non-truncating variants account for 30-40% of cases, and their impact on essential DDX41 functions remains unknown. We utilized a genetic complementation assay to assess the functionality of 10 recurrent germline non-truncating variants of DDX41. All variants restored viability to Ddx41 -deficient hematopoietic progenitor cells at exogenous expression levels. In contrast, the hotspot mutant p.R525H, which is somatically acquired at disease onset in >50% of patients, failed to restore viability. CRISPR-based modeling in cell lines and mice revealed heterogeneity: some variants were non-functional at endogenous expression levels whereas others maintained complete functionality, supporting normal cell proliferation and even lifelong hematopoiesis in a homozygous setting. Notably, co-expression of p.R525H with some variants caused impaired hematopoietic progenitor cell viability, indicating a dominant-negative effect of p.R525H. In contrast, other variants, all classified as variants of unknown significance, were unaffected by the presence of p.R525H. A screen of 100 disease-associated variants confirmed that many non-truncating germline variants are susceptible to p.R525H-mediated dominant-negative effects, whereas wild-type DDX41 is not. These findings indicate that DDX41 variant curation is complicated by variable effects on functionality and variant-specific interactions with somatically-acquired DDX41 mutations. The dominant-negative effect of p.R525H provides a mechanistic basis for the conclusion of recent patient cohort analyses that co-occurrence with a somatic hotspot mutation is a reliable indicator of DDX41-driven disease in carriers of non-truncating variants.

Abstract Heterozygous germline variants of DDX41 occur in 2-4% of adult Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) cases, making them the most prevalent cause of inherited predisposition to these diseases. Homozygous deleterious variants are never observed in the germline, and loss of both DDX41 alleles causes apoptosis of proliferative cells, consistent with essentiality. Most disease-associated germline variants cause loss of full-length protein expression (truncating). However, 30-40% are missense (non-truncating) and their role in disease risk compared to truncating variants remains in question. Previous reports have shown that both truncating and non-truncating Pathogenic/Likely Pathogenic (P/LP) variants (defined by ACMG scoring) are associated with DDX41-specific disease features, including co-mutation of the contralateral allele of DDX41. In contrast, variants of unknown significance (VUS) are less associated with these features. Acquired co-mutations of DDX41, of which p.R525H accounts for 50-70%, are restricted to DDX41-mutated patients and are always missense. Our previous studies indicated that p.R525H lacks the essential DDX41 function and cannot support hematopoietic progenitor proliferation. However, the frequency with which this mutation occurs and the absence of acquired truncating mutations suggest that p.R525H has a specific effect on DDX41 function that is distinct from complete loss and promotes pathogenesis specifically in the context of existing P/LP DDX41 mutation. To understand how germline non-truncating variants predispose to disease, we characterized the effect of six recurrent non-truncating P/LP variants (p.G173R, p.R219H, p.P258L, p.Y259C, p.K331del, and p.I396T) and three VUS (p.K9E, p.Y33H, and p.M155H) on DDX41 function. We utilized a genetic complementation approach in murine lineage-negative (Linneg) bone marrow cells with inducible deletion of DDX41. We found that all non-truncating P/LP and VUS forms of DDX41 could fully rescue the colony forming potential of Ddx41-deficient cells to a similar level as wild-type protein, whereas the p.R525H variant completely failed to rescue. To determine the effect of the non-truncating variants on DDX41 function at endogenous expression levels, we utilized CRISPR to modify the DDX41 alleles in MOLM13 human AML cells. We produced viable, proliferating cell lines with homozygous mutations for all 3 VUS and 4/6 P/LP variants. For p.P258L and p.K331del, like p.R525H, we could not produce homozygous mutant cell lines despite screening hundreds of clones and finding several with heterozygosity. To further study the P/LP variants that were functional in the cell line, we generated C57Bl/6 mouse lines harboring p.G173R, p.Y259C, and p.I396T mutations in the endogenous Ddx41 alleles. Heterozygous mutant mice were phenotypically normal in all three strains. However, we could not obtain homozygous mutant mice for p.G173R or p.Y259C, indicating these cannot support embryonic/fetal development. In contrast, p.I396T-homozygous mutant mice were born at Mendelian ratios and were phenotypically normal until at least 15 months of age. Since p.R525H mutation is common in patients with non-truncating P/LP variants, we sought to model the combined effect of these mutations. We utilized the Linneg cells with inducible knockout of Ddx41, co-expressed each non-truncating variant with p.R525H by viral transduction, and monitored the colony formation potential. Remarkably, cultures expressing P/LP variants or M155I combined with p.R525H had dramatically reduced colony formation, while wild-type DDX41 and the other 2 VUS supported normal colony formation when co-expressed with p.R525H. Thus, we conclude p.R525H has a dominant-negative effect on certain non-truncating DDX41 variants. This is consistent with the emerging idea that the presence of an acquired hotspot DDX41 co-mutation is sufficient to classify a non-truncating DDX41 variant as pathogenic.Our results demonstrate that many DDX41 missense variants retain protein function when overexpressed. However, at endogenous expression levels, some P/LP variants are unable to support cell proliferation, indicative of hypomorphic effects on protein function. In patients, acquisition of the p.R525H mutation in the contralateral allele disrupts the functionality of P/LP variants through dominant-negative effects, leading to hematopoietic progenitor defects that are hallmark of the DDX41 pathogenesis.

Background: We evaluated imaging features suggestive of neurodegeneration within the brainstem and upper cervical spinal cord (UCSC) in non-progressive multiple sclerosis (MS). Methods: Standardized 3-Tesla three-dimensional brain magnetic resonance imaging (MRI) studies were prospectively acquired. Rates of change in volume, surface texture, curvature were quantified at the pons and medulla-UCSC. Whole and regional brain volumes and T2-weighted lesion volumes were also quantified. Independent regression models were constructed to evaluate differences between those of Black or African ancestry (B/AA) and European ancestry (EA) with non-progressive MS. Results: 209 people with MS (pwMS) having at least two MRI studies, 29% possessing 3–6 timepoints, resulted in 487 scans for analysis. Median follow-up time between MRI timepoints was 1.33 (25th–75th percentile: 0.51–1.98) years. Of 183 non-progressive pwMS, 88 and 95 self-reported being B/AA and EA, respectively. Non-progressive pwMS demonstrated greater rates of decline in pontine volume ( p < 0.0001) in B/AA and in medulla-UCSC volume ( p < 0.0001) for EA pwMS. Longitudinal surface texture and curvature changes suggesting reduced tissue integrity were observed at the ventral medulla-UCSC ( p < 0.001), dorsal pons ( p < 0.0001) and dorsal medulla ( p < 0.0001) but not the ventral pons ( p = 0.92) between groups. Conclusions: Selectively vulnerable regions within the brainstem-UCSC may allow for more personalized approaches to disease surveillance and management.

Abstract While it is known that nuclear factor κ-B (NF-κB) signaling within macrophages regulates expression of downstream targets that can mediate either pro- or antitumor functions, the mechanisms that define predominant characteristics are not well understood. As macrophages are present in the tumor microenvironment of diverse tumor types, their characteristics have the potential to either support or hinder tumor growth and metastatic spread. Therefore, a method to shift the balance of tumor associated macrophages from pro- to antitumor functions could potentially target tumor cells directly and also recruit and activate other immune cells, resulting in significant therapeutic benefit. Our studies are designed to gain an understanding of how to use modulation of NF-κB signaling as the “pinch point” to generate antitumor phenotypes and determine how to achieve this modulation in a patient. We are using immortalized bone marrow-derived macrophages or ex vivo tumor-associated macrophages in cell culture approaches and inducible transgenic mice in in vivo approaches to investigate the mechanisms by which NF-κB defines macrophage characteristics. Interactions with tumor cells are also being investigated using co-culture and murine models. Our data suggests that high levels of NFκ-B activity in macrophages induce both direct tumor cell killing and immune stimulating responses. We are testing two different translational approaches. The first is liposomal muramyl tripeptide ethanolamine that acts as an immune stimulant by increasing NF-κB activity in macrophages. The second is preferential delivery of siRNA against the inhibitor of NFκ-B (IκB-alpha) to tumor-associated macrophages using polymeric nanoparticles. We hope to better understand the mechanisms by which NFκ-B regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that could be effective across a wide spectrum of solid tumors and metastatic disease. Citation Format: Esther Liu, Alyssa A. Hoover, Whitney R. Harris, Whitney Barham, Oleg Tikhomirov, Dineo Khabele, Zahra Mirafzahli, Todd D. Giorgio, Andrew J. Wilson, Fiona E. Yull. Modulation of NFκ-B signaling to optimize antitumor characteristics in macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4711.