Abstract 12653: The Therapy-related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-ischemic Heart Failure in a Murine Model

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Yoshimitsu Yura ◽  
Emiri Miura-Yura ◽  
Kenneth Walsh
Keyword(s):  
Stem Cells ◽  
Angiotensin Ii ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Cancer Patients ◽  
Nlrp3 Inflammasome ◽  
Cardiac Dysfunction ◽  
Hematopoietic Stem ◽  
Cardiac Stress ◽  
Clonal Hematopoiesis

Background: Therapy-related clonal hematopoiesis in cancer patients is typically associated with somatic mutations in hematopoietic cell genes that encode regulators of the DNA-damage response (DDR) pathway. The Protein Phosphatase Mg2+/Mn2+ Dependent 1D ( PPM1D ) gene is the most frequently mutated DDR gene associated with therapy-related clonal hematopoiesis. While epidemiological evidence suggests an association between therapy-related clonal hematopoiesis and cardiovascular disease in cancer patients, causal and mechanistic relationships have never been evaluated in an experimental system. Methods: To test whether hematopoietic cell mutations in PPM1D can increase the susceptibility to cardiac stress, we evaluated cardiac dysfunction in response to angiotensin II infusion in a mouse model where clonal-hematopoiesis-associated mutations in Ppm1d were produced by CRISPR-Cas9 technology. Results: Mice transplanted with hematopoietic stem cells containing clinically relevant mutations in exon 6 of Ppm1d exhibited augmented cardiac remodeling following the continuous infusion of angiotensin II. Ppm1d -mutated macrophages showed impairments in the DDR pathway and had an augmented proinflammatory profile. Mice transplanted with Ppm1d mutated cells exhibited elevated IL-1β in the stressed myocardium, and bone marrow derived macrophages produced more IL-1β in response to LPS stimulation. The administration of an NLRP3 inflammasome inhibitor to mice reversed the cardiac phenotype induced by the Ppm1d -mutated hematopoietic stem cells under conditions of Angiotensin II-induced stress. Conclusions: A mouse model of Ppm1d -mediated clonal hematopoiesis was more susceptible to cardiac stress following of angiotensin II infusion. Mechanistically, disruption of the DDR pathway led to elevations in inflammatory cytokine production, and the NLRP3 inflammasome was shown to be essential for this augmented cardiac stress response. These data indicate that therapy-related clonal hematopoiesis involving mutations in PPM1D could contribute to the cardiac dysfunction observed in cancer survivors.

scholarly journals The Cancer Therapy-Related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-Ischemic Heart Failure in Mice

2021 ◽  
Author(s):  
Yoshimitsu Yura ◽  
Emiri Miura-Yura ◽  
Yasufumi Katanasaka ◽  
Kyung-Duk Min ◽  
Nicholas W Chavkin ◽  
...  
Keyword(s):  
Heart Failure ◽  
Stem Cells ◽  
Dna Damage ◽  
Cancer Therapy ◽  
Mouse Model ◽  
Cardiac Dysfunction ◽  
Ischemic Heart Failure ◽  
Hematopoietic Stem ◽  
Cardiac Stress ◽  
Clonal Hematopoiesis

Rationale: Cancer therapy can be associated with short- and long-term cardiac dysfunction. Cancer patients often exhibit therapy-related clonal hematopoiesis (t-CH), an aggressive form of clonal hematopoiesis that can result from somatic mutations in genes encoding regulators of the DNA-damage response (DDR) pathway. Gain-of-function mutations in exon 6 the protein phosphatase Mg2+/Mn2+ dependent 1D (PPM1D) gene are the most frequently mutated DDR gene associated with t-CH. Whether t-CH can contribute to cardiac dysfunction is unknown. Objective: We evaluated the causal and mechanistic relationships between Ppm1d-mediated t-CH and non-ischemic heart failure in an experimental system. Methods and Results: To test whether gain-of-function hematopoietic cell mutations in Ppm1d can increase the susceptibility to cardiac stress, we evaluated cardiac dysfunction in a mouse model where clonal hematopoiesis-associated mutations in exon 6 of Ppm1d were produced by CRISPR-Cas9 technology. Mice transplanted with hematopoietic stem cells containing the mutated Ppm1d gene exhibited augmented cardiac remodeling following the continuous infusion of angiotensin II (AngII). Ppm1d-mutant macrophages were impaired in DDR pathway activation and displayed greater DNA damage, higher reactive oxygen species generation and an augmented proinflammatory profile with elevations in IL-1β and IL-18. The administration of an NLRP3 inflammasome inhibitor to mice reversed the cardiac phenotype induced by the Ppm1d-mutated hematopoietic stem cells under conditions of AngII-induced stress. Conclusions: A mouse model of Ppm1d-mediated t-CH was more susceptible to cardiac stress. Mechanistically, disruption of the DDR pathway led to elevations in inflammatory cytokine production, and the NLRP3 inflammasome was shown to be essential for this augmented cardiac stress response. These data indicate that t-CH involving activating mutations in PPM1D can contribute to the cardiac dysfunction observed in cancer survivors, and that anti-inflammatory therapy may have utility in treating this condition.

scholarly journals Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

2021 ◽  
Vol 9 ◽  
Author(s):  
Wanbo Tang ◽  
Jian He ◽  
Tao Huang ◽  
Zhijie Bai ◽  
Chaojie Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Hematopoietic Stem ◽  
Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

Neuroprotective effect of adult hematopoietic stem cells in a mouse model of motoneuron degeneration

2007 ◽  
Vol 26 (2) ◽  
pp. 408-418 ◽  
Author(s):  
Carmen Cabanes ◽  
Sonia Bonilla ◽  
Lucía Tabares ◽  
Salvador Martínez
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Neuroprotective Effect ◽  
Hematopoietic Stem ◽  
Motoneuron Degeneration

Clonal hematopoiesis by SLIT1-mutated hematopoietic stem cells due to a breakdown of the autocrine loop involving Slit1 in acquired aplastic anemia

Leukemia ◽  
2019 ◽  
Vol 33 (11) ◽  
pp. 2732-2766
Author(s):  
Kohei Hosokawa ◽  
Hiroki Mizumaki ◽  
Mahmoud I. Elbadry ◽  
Chizuru Saito ◽  
J. Luis Espinoza ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Stem ◽  
Autocrine Loop ◽  
Clonal Hematopoiesis

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3737-3747 ◽  
Author(s):  
Dirk Heckl ◽  
Daniel C. Wicke ◽  
Martijn H. Brugman ◽  
Johann Meyer ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Rare Hematopoietic Subclones Harboring Leukemogenic TP53 Mutations Are Detectable Via Error-Corrected Sequencing in Healthy Elderly Individuals

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2907-2907
Author(s):  
Andrew Young ◽  
Terrence Neal Wong ◽  
Timothy J Ley ◽  
Daniel C. Link ◽  
Todd E Druley
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Error Rate ◽  
X Inactivation ◽  
Research Network ◽  
Hematopoietic Stem ◽  
Elderly Individuals ◽  
Tp53 Mutations ◽  
Clonal Hematopoiesis ◽  
Healthy Elderly

Abstract Acute myeloid leukemia (AML) is an oligoclonal disease marked by specific somatic genomic alterations. While the leukemia-associated mutations and rearrangements differ between individual cases, the set of recurrently mutated genes is now largely known (Cancer Genome Atlas Research Network, NEJM 2013). Current evidence supports a model of leukemogenesis, by which leukemia-associated mutations are acquired sequentially over time in hematopoietic stem cells (HSCs). Furthermore, “pre-leukemic” HSCs, which contain only a subset of the mutations found in the dominant clone, are detectable at diagnosis (Corces-Zimmerman MR, et al., PNAS 2014; Shlush LI, et al., Nature 2014). Despite these observations, the effect of these mutations, when they first arise in healthy HSCs, is largely unknown. It is likely that these early mutations endow a selective growth advantage to the HSC resulting in detectable clonal hematopoiesis without immediately causing overt leukemia. As expected, there is evidence from studies of X-inactivation skewing that clonal hematopoiesis exists in the blood of healthy elderly individuals (Busque L, et al. Blood 2009). In a separate study, hematopoietic X-inactivation skewing in elderly individuals was associated with TET2 mutations in 10/182 cases (Busque L, et al. Nat Genet 2012). This study was only capable of detecting insertions or deletions due to the high (~1%) substitution error rate of conventional next-generation sequencing (NGS) and likely underreported the prevalence of clonal hematopoiesis harboring putative driver mutations in TET2. To further study the role of leukemia-associated single nucleotide variants in healthy hematopoiesis, we applied our validated method for targeted error-corrected sequencing (ECS). ECS uses random, single molecule indexing to overcome the inherent error rate of NGS by establishing “read families” from multiple reads generated from each unique index (Schmitt MW, et al. PNAS 2012, Kinde I, et al., PNAS 2012). A dilution series of two independent mutations with technical replicates demonstrated that ECS enables the quantitative identification of variants as rare as 1:10,000 molecules. We applied ECS to identify and quantify leukemia-associated subclones harboring mutations in TP53 exons 4-7, which is where the majority of cancer-related mutations in TP53 have been described. ECS libraries were generated from blood samples drawn from 20 healthy elderly individuals (average 75 years old). Sample multiplexing for sequencing was accomplished by tagging PCR amplicons, generated from each individual, with a different oligonucleotide barcode during library preparation. The resulting individual ECS libraries were then multiplexed and sequenced on one lane of the Illumina HiSeq 2500 platform. Sequence reads originating from the same randomly indexed molecule are aligned to each other to generate read families. First, at every position, the bases called by each sequence read are compared and a consensus base is called if there is ≥90% agreement between the reads. If there is less than 90% agreement, the consensus base is called an N. Sequencing errors are thus removed since they are not shared between different reads within a read family. Second, an error corrected consensus sequence (ECCS) is discarded if <90% of bases across a paired-end read are non-N. ECCSs are locally aligned to hg19/GRCh30 using bowtie2. We identified rare subclonal hematopoiesis harboring TP53 mutations in 9 of 20 healthy individuals at variant allele frequencies (VAF) between 1:10,000 and 1:270. Of the 13 identified mutations, 12 were coding or splicing mutations and 10 had been previously identified as leukemia-associated in the Catalog of Somatic Mutations in Cancer. We validated three independent variants with droplet digital PCR and measured nearly identical VAFs at each loci. These findings suggest that potentially oncogenic mutation in hematopoietic stem cells is a stochastic process and rare subclonal hematopoiesis is a common occurrence in healthy aged individuals, which is consistent with the observation that de novo AML primarily occurs in the elderly. Ongoing studies are applying ECS to determine the prevalence of rare subclonal mutation in other recurrently mutated AML genes. These studies will help further elucidate the natural history of leukemogenesis and may enable the accurate detection of individuals at risk for developing cancer. Disclosures No relevant conflicts of interest to declare.

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