Genomic context and TP53 allele frequency define clinical outcomes in TP53-mutated myelodysplastic syndromes

TP53 mutations are associated with adverse outcomes and shorter response to hypomethylating agents (HMAs) in myelodysplastic syndrome (MDS). Limited data have evaluated the impact of the type, number, and patterns of TP53 mutations in response outcomes and prognosis of MDS. We evaluated the clinicopathologic characteristics, outcomes, and response to therapy of 261 patients with MDS and TP53 mutations. Median age was 68 years (range, 18-80 years). A total of 217 patients (83%) had a complex karyotype. TP53 mutations were detected at a median variant allele frequency (VAF) of 0.39 (range, 0.01-0.94). TP53 deletion was associated with lower overall response rate (ORR) (odds ratio, 0.3; P = .021), and lower TP53 VAF correlated with higher ORR to HMAs. Increase in TP53 VAF at the time of transformation was observed in 13 patients (61%), and previously undetectable mutations were observed in 15 patients (65%). TP53 VAF was associated with worse prognosis (hazard ratio, 1.02 per 1% VAF increase; 95% confidence interval, 1.01-1.03; P \u3c .001). Integration of TP53 VAF and karyotypic complexity identified prognostic subgroups within TP53-mutant MDS. We developed a multivariable model for overall survival that included the revised International Prognostic Scoring System (IPSS-R) categories and TP53 VAF. Total score for each patient was calculated as follows: VAF TP53 + 13 × IPSS-R blast score + 16 × IPSS-R cytogenetic score + 28 × IPSS-R hemoglobin score + 46 × IPSS-R platelet score. Use of this model identified 4 prognostic subgroups with median survival times of not reached, 42.2, 21.9, and 9.2 months. These data suggest that outcomes of patients with TP53-mutated MDS are heterogeneous and that transformation may be driven not only by TP53 but also by other factors

Hematopoiesis under telomere attrition at the single-cell resolution

The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells’ self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells’ skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance

Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages.

Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis

SP-D treatment does not alter <i>C</i>. <i>burnetii</i> stimulated transcriptional patterns in MH-S cells.

<p>MH-S cells were infected with PBS or SP-D treated <i>C</i>. <i>burnetii</i> RSA439 (phase II), RSA493 (phase I) or treated with <i>E</i>. <i>coli</i> LPS for 0, 4, and 8 hours. RNA was extracted as described and applied to real-time PCR. Data represent the mean ± standard deviation of fold change in RNA expression of select genes compared to expression of host cell <i>Gapdh</i>, n = 3. RSA439 and LPS data are representative of three independent experiments, RSA493 data are representative of two independent experiments. Student t-tests compare each time and condition to its corresponding 0 time point, all RSA439 and RSA439 + SP-D were significantly up-regulated * p<0.05. There was no significant difference in expression between untreated and SP-D treated <i>C</i>. <i>burnetii</i> RSA439 as determined by student t-tests.</p

SP-D is not bactericidal towards <i>C</i>. burnetii.

<p>(A) <i>E</i>. <i>coli</i> were treated with or without SP-D for one hour at 37°C followed by viable counts on LB. Data displayed are mean ± standard error of three independent experiments. Student t-test compares SP-D to PBS treated <i>E</i>. <i>coli</i>. * p<0.05. (B) <i>C</i>. <i>burnetii</i> were treated with 0 (triangles), 5 (squares) or 10 mg/ml (squares) SP-D overnight and transferred to ACCM-2. Every 2 days genome equivalents (GE) were calculated by real-time PCR. Data displayed are mean ± standard deviation of GE/mL, representative of three independent experiments.</p

SP-D treatment results in a decrease in infectivity.

<p>ACCM-2 or L-929 passaged <i>C</i>. <i>burnetii</i> were stained with CSFE and treated overnight with SP-D or the equivalent volume of buffer alone (PBS). MH-S cells were infected at MOI 100 for 4 or 8 hours and fixed. MH-S nuclei were stained with Hoescht, the number of infected cells and bacteria was quantified via confocal microscopy, and the infectivity index was calculated (bound and internalized <i>C</i>. <i>burnetii</i>). Data are displayed as mean ± standard deviation of infectivity from one representative of 5 experiments, n = 3. Student t-tests compare SP-D to PBS treated groups. *, p<0.05, ***, p<0.0005.</p

SP-D binds to <i>C</i>. <i>burnetii</i> in a calcium dependent fashion that can be inhibited by monosaccride.

<p>SP-D was incubated with <i>C</i>. <i>burnetii</i> or <i>E</i>. <i>coli</i> with or without EDTA or galactose, in PBS + CaCl₂ overnight at 37°C. Bacterial suspensions were then washed 3x with PBS by centrifugation. All samples were subjected to SDS-PAGE followed by blotting to detect SP-D that co-sedimented with the bacteria. SP-D was loaded as a positive control (left lane each blot labeled SP-D). Representative results are shown from 3 independent experiments.</p