Effects of PARP Inhibitor Therapy on p53-Deficient Hematopoietic Stem and Progenitor Cell Fitness

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors are an important new class of anti-cancer therapies. Therapy-related myeloid neoplasia (tMN) has been reported following PARPi therapy and is associated with adverse outcomes. We have previously shown, in retrospective data, that prior chemotherapy increases the incidence of clonal hematopoiesis (CH), especially in DNA damage response (DDR) pathway genes including TP53, PPM1D, and CHEK2 and is associated with progression to tMN. In particular, patients who receive PARPi therapy are more likely to have CH compared to other therapies or untreated patients. In the IMPACT study of CH in 10,156 cancer patients, exposure to PARPi were more likely to have CH (33%) compared to untreated patients (16%). This was particularly pronounced for DDR gene mutations, with 25% of PARPi treated patients with DDR CH compared to 2% of untreated patients. In multivariate analysis accounting for demographics and exposure to other chemotherapy or radiation therapy, exposure to PARPi conferred an increased risk of DDR CH (OR = 3.6, 95% CI 1.5-8.5, p = 0.004). From these data, we hypothesize that mutations in DDR pathway genes provide a fitness advantage to hematopoietic stem/progenitor cells (HSPCs) following PARPi treatment, leading to clonal hematopoiesis. A major limitation, however of our previous work in retrospective clinical samples, is the inability to completely adjust for the confounding effect of prior exposure to cytotoxic therapy (in particular platinum therapies) and germline BRCA1/2 mutations; both which have been shown or hypothesized to increase the risk of tMN. To test whether PARPi exposure might provide a fitness advantage to HSPCs independent of prior exposure to other therapies, we first examined the response of CRISPR-gene edited TP53-/- MOLM13 cells to the PARPi Olaparib and, as a control, Cisplatin. As expected, TP53-/- cells had increased resistance to both agents, though the response was much more pronounced in Cisplatin-treated cells (Figure 1A,B). Next, we implemented a mouse model of TP53-mutant clonal hematopoiesis, by generating mixed bone marrow chimeras transplanted with a 1:9 ratio of wildtype (CD45.1) to TP53 R172H+/- (CD45.2) cells. The "baseline" contribution of TP53 R172H+/- (CD45.2) cells to peripheral blood leukocytes 8 weeks after transplantation was determined by flow cytometry. Mice were then randomized into the following three cohorts: 1) Cisplatin (6mg/kg on days 1, 8, and 15); 2) Olaparib (50mg/kg daily for 3 weeks); and 3) vehicle alone. Peripheral blood chimerism was assessed 3, 9, and 12 weeks after initiating treatment. In addition, the contribution of TP53 R172H+/- to lineage -Sca1 +Kit + (LSK) cells in the bone marrow was determined. Cisplatin treatment resulted in a significant increase in the contribution of TP53 R172H+ to peripheral blood total leukocytes, granulocytes, and bone marrow LSK cells (Figure 1C-E). In contrast, Olaparib treated mice showed no change in CD45 chimerism. From these results we conclude that p53-deficiency does not confer a strong fitness advantage to mouse HSPCs in response to PARPi treatment. This suggests that the strong association observed between prior PARPi therapy, CH and tMN in clinical cohorts may in part be due to the confounding effects of prior (often heavy) exposure to platinum-based therapy. However, the majority of patients receiving PARPi have germline heterozygous BRCA1/2 mutations that could be contributing to their hematopoietic response to PARPi therapy. Experiments are underway to test this possibility by analyzing mixed bone marrow chimeras carrying heterozygous mutations of both Brca1 and Trp53. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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CIRCULATING microRNA EXPRESSION IN MYELODYSPLASTIC SYNDROME

Liječnički vjesnik ◽

10.26800/lv-141-7-8-30 ◽

2019 ◽

Vol 141 (7-8) ◽

pp. 233-237

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Peripheral Blood ◽

Scoring System ◽

Molecular Mechanisms ◽

International Prognostic Scoring System ◽

Circulating Microrna ◽

Hematopoietic Stem ◽

The Impact

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and cytopenia in peripheral blood, where about a third of patients may develop acute myeloid leukemia (AML). The diagnosis of MDS requires the analysis of peripheral blood and bone marrow. Depending on the percentage of blasts in the bone marrow, the number of cytopenias and cytogenetic abnormalities, determination of the prognostic indices is possible (IPSS – „International Prognostic Scoring System“, R-IPSS-„Revised International Prognostic Scoring System“, WPSS – „WHO Prognostic Scoring System“). Until today, numerous studies have been conducted on the molecular mechanisms and epigenetic pathways in myelodysplastic syndrome, and their prognostic and therapeutic importance, but there are few studies analyzing the importance of microRNAs (miRNAs) in MDS. In the last few years, there have been numerous results on the impact of aberrant miRNA expression in malignant disorders where the miRNA represent tumor suppressor genes or oncogenes. Several miRNAs have been recognized as diagnostic and prognostic parameters and possible therapeutic targets. In this paper, we present the overview of recent results on the role of miRNA in MDS.

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Reversing Clonal Hematopoiesis and Associated Atherosclerotic Disease By Targeted Antibody-Drug-Conjugate (ADC) Conditioning and Transplant

Blood ◽

10.1182/blood-2020-143176 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-35

Author(s):

Karin Gustafsson ◽

Catherine S Rhee ◽

Elizabeth W Scadden ◽

Vanessa Frodermann ◽

Rahul Palchaudhuri ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Peripheral Blood ◽

Publicly Traded ◽

Antibody Drug Conjugate ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

The Past ◽

Antibody Drug

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recently, age-related clonal hematopoiesis (CH) has been recognized as a risk factor for CVD of comparable magnitude to smoking, hypertension and hypercholesteremia. While these other risk factors can be mitigated by pharmacological intervention or lifestyle changes, there are no such strategies in place for CH. As CH is initiated by mutations in hematopoietic stem cells (HSCs), a hematopoietic stem cell transplantat (HSCT) could serve as a curative therapy. However, stem cell transplantation is associated with significant toxicity due in part from current conditioning regimens. There is also no evidence that depletion of the disease-driving clones impacts established atherosclerosis. We developed an antibody drug conjugate (ADC) targeting murine CD45. In the context of stem cell transplantation, the CD45-ADC efficiently depletes endogenous HSCs as well as mature leukocytes while enabling rapid engraftment of an infused stem cell graft. In addition, the CD45-ADCs are not based on broad-acting genotoxic agents that lead to long-lasting health risks. We decided to test if CD45-ADC and HSCT could halt atherosclerosis progression through elimination Tet2 knockout HSCs and their disease propagating myeloid progeny. To model CH associated atherosclerosis, LDLR knockout mice were transplanted with 20% CFP labeled wild-type (WT) or Tet2 knockout bone marrow. A single dose of isotype- or CD45-ADC was delivered after 6 weeks of atherosclerosis development and was followed by an infusion of WT CD45.1 bone marrow. As has been reported before, we observed in the isotype-ADC treated animals that Tet2 deficiency leads to a competitive advantage over WT cells. Tet2 knockout cells contributed to peripheral blood chimerism at successively increasing levels and mice harboring the knockout graft showed a significant expansion of their HSC population. Despite their obvious advantage, Tet2 deficient HSC were as efficiently depleted as their WT counterparts upon CD45-ADC and HSCT. Peripheral blood and bone marrow chimerism were similar in WT and Tet2 knockout hosts and the expanded HSC pool was successfully curbed 6 weeks following the intervention. More importantly, CD45-ADC also depleted cells in the atherosclerotic plaques as efficiently as in blood in both WT and Tet2 mutant recipients. This resulted in a significant reduction of myeloid cell infiltration in CD45-ADC conditioned and transplanted knockout hosts and ultimately lead to drastically reduced plaque size in these animals. In conclusion, these data demonstrate that CD45-ADC and HSCT efficiently replaces the disease driving myeloid cells in the atherosclerosis plaques leading to an overall reduction in disease burden. CD45-ADC and transplantation may thus offer a novel therapy for CH and its associated morbidities. Disclosures Palchaudhuri: Magenta Therapeutics: Current Employment. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Proctor:Magenta Therapeutics: Current Employment. Gillard:Magenta Therapeutics: Current Employment. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

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Mosaic Tetraploidy and Transient Mutations in the GFI1 Gene in a Patient with Severe Chronic Neutropenia.

Blood ◽

10.1182/blood.v108.11.1281.1281 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1281-1281

Author(s):

Andrew A.G. Aprikyan ◽

Jessica C. Hochberg ◽

Patricia M. Miron ◽

Beverly N. Hay ◽

Bruce A. Woda ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Mutational Analysis ◽

Genetic Defect ◽

Flow Cytometric Analysis ◽

Chromosome 21 ◽

Embryonic Cells ◽

Hematopoietic Stem ◽

Increased Risk ◽

History Of

Abstract Severe congenital neutropenia (SCN), also known as Kostmann disease, is a rare disorder characterized by peripheral blood agranulocytosis and maturation arrest of neutrophils within the bone marrow at the promyelocytic stage. Patients usually present with an absolute neutrophil count below 0.2 x 109/L and severe infections, with the typical onset of symptoms during infancy. Most patients respond to treatment with granulocyte colony-stimulating factor, but retain an increased risk for death from sepsis and for development of myelodysplastic syndrome and acute myeloid leukemia (MDS/AML). Several reports have described karyotype abnormalities associated with this syndrome, most typically clonal loss of chromosome 7 or abnormalities of chromosome 21 associated with transformation to MDS/AML. The etiology of these abnormalities and their role in progression to MDS/AML remains unclear. Here we present a case of a 15 year-old boy with SCN, lymphopenia, and persistent tetraploid mosaicism in the bone marrow and in subpopulations of peripheral blood leukocytes. His phenotype, including growth and development, was otherwise normal. The patient’s father, who had mild neutropenia and a history of monoclonal gammopathy, also showed a tetraploid subpopulation in his bone marrow. Flow cytometric analysis of the DNA content of CD45+ nucleated cells in the patient’s peripheral blood showed that 20% of CD13+ granulocytes, and 15% of CD14+ monocytes were tetraploid. The lifelong persistence of the mosaicism and the similar finding in the patient’s father suggest that it represents a congenital propensity towards acquiring tetraploidy. However, cytogenetic evaluation of the patient’s skin fibroblasts detected no tetraploidy, indicating failure of the putative tetraploid embryonic cells to contribute to that lineage. The fact that the patient’s father also had a population of tetraploid cells in his bone marrow and a history of neutropenia suggests a common underlying genetic cause for both conditions in this kindred. Mutational analysis revealed that the ELA2 gene sequence was normal, but the GFI1 gene exhibited transient, simultaneous appearance of two novel mutations: a missense substitution in exon 1 and a single base change in the promoter region, at a putative binding site for the myeloid-specific transcription factor MZF1. The GFI1 mutations were repeatedly identified in a DNA sample obtained in 2003, but not in 2001, 2002, or 2006 samples. Sequencing of DNA obtained from buccal swabs of the patient and his parents also revealed no GFI1 mutation. The transient appearance may represent the emergence and subsequent loss of a clone of hematopoietic stem or progenitor cells with GFI1 mutations. This hypothesis is supported by a recent report demonstrating that GFI1 mutation results in premature apoptosis of myeloid cells, similar to apoptosis-inducing mutations in the neutrophil elastase gene ELA2. The fact that GFI1 mutations were identified in a DNA sample from one year, but not from others, raises the possibility that GFI1 mutations reported in other neutropenic patients may also be transient. We speculate that an underlying genetic defect, inherited in an autosomal dominant pattern, leads to both disordered mitosis and leukopenia in this kindred.

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High Prevalence of PPM1D Mutations in Therapy-Related AML/MDS Is Due to Context-Specific Clonal Hematopoiesis

Blood ◽

10.1182/blood-2018-99-115007 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 746-746

Author(s):

Joanne Hsu ◽

Tajhal Dayaram ◽

Ayala Tovy ◽

Etienne De Braekeleer ◽

Mira Jeong ◽

...

Keyword(s):

Dna Damage ◽

Peripheral Blood ◽

Somatic Mutations ◽

De Novo ◽

Prognostic Significance ◽

Specific Treatment ◽

Chemotherapeutic Agents ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Fitness Advantage

Abstract Clonal hematopoiesis (CH) is a condition in which individual hematopoietic stem cells (HSCs) acquire a fitness advantage and contribute disproportionately to peripheral blood production. Somatic mutations in around 20 genes are recurrently associated with CH, with truncating mutations in PPM1D being among the most common. PPM1D encodes the WIP1 protein (Wildtype p53-Induced Phosphatase 1), which is upregulated by p53 during DNA damage and acts homeostatically to downregulate the DNA damage response (DDR). More recently, PPM1D mutations have been observed in the blood of individuals who had previously been exposed to chemotherapeutic agents for prior malignancies. The effect of PPM1D truncating mutations on hematopoiesis remains unclear, as does the precise mechanism by which PPM1D mutations confer a fitness advantage to hematopoietic stem and progenitor cells in CH. To explore the prevalence and features of PPM1D mutations in a focused subset of patients with prior chemotherapy exposure, we screened for PPM1D mutations in 156 patients with t-AML (n=77) and t-MDS (n=79) by targeted-capture deep sequencing of PPM1D and 295 cancer genes. Truncating mutations in PPM1D were detected in 31/156 cases (20%) with a mean VAF of 0.105 (range 0.02-0.48), making it the second most commonly mutated gene in t-AML/MDS after TP53 (29%). In contrast, PPM1D mutations were detected in only 0.5% of a matched de novo MDS/AML cohort, confirming a specific enrichment in therapy-related cases (Fig 1a). Notably, PPM1D-mutant CH was specifically associated with prior exposure to platinum agents (p=0.004) and etoposide (p=0.021). To investigate the mechanisms behind this clinical association, we created PPM1D truncating mutations in multiple cell lines using CRISPR-Cas9. The truncated WIP1 protein was highly stabilized, leading to a net hyperactive effect on the dephosphorylation of DDR pathway members including phospho-p53 and γH2AX. We next asked whether this translates to chemoresistance, by comparing the sensitivities of PPM1D-mutant and wild-type (WT) cells to various agents. PPM1D mutants were resistant to DNA-damaging agents such as cisplatin, etoposide and doxorubicin, but not to vincristine, a microtubule inhibitor, indicating enhanced survival in specific contexts. We then asked whether this conferred an advantage, by competing PPM1D-mutant and WT cells in vitro with multiple exposures. PPM1D mutants did not have an advantage at baseline or in the context of vincristine, but significantly outcompeted their WT counterparts when exposed to cisplatin, etoposide, and doxorubicin. Annexin V staining revealed that PPM1D mutants exhibit diminished apoptosis with DDR-inducing agents, explaining most of their competitive advantage. Co-treatment with a PPM1D inhibitor reversed this gain and may have clinical implications. To understand the in vivo parameters that impact PPM1D-mutant cell fitness, we next generated a novel Ppm1d truncated knock-in mouse model. Characterization of baseline hematopoiesis in the Ppm1d mutant mouse revealed no appreciable differences in lineage composition or proportion of hematopoietic progenitors. To analyze the factors that impact clonal evolution, we competed Ppm1d-mutant and WT cells in specific proportions in bone marrow transplantation. In the context of stress from transplantation alone, Ppm1d-mutant and WT HSCs remained at the same proportions as the initial transplant. Similarly, serial transplantation did not alter their relative contributions to peripheral blood production. In contrast, competitively transplanted mice treated with cisplatin demonstrated a striking selection for Ppm1d mutants in both the peripheral blood (Fig 1b) and LT-HSCs, suggesting that Ppm1d-mutant clones achieve greater fitness and gain a selective advantage under specific extrinsic stressors. This differs from CH-associated mutations in DNMT3A and TET2, where intrinsic characteristics such as enhanced self-renewal appear sufficient to drive clonal expansion. Broadly, this study highlights the significance of context-specificity underlying CH of different somatic mutations. Furthermore, these findings establish the prognostic significance of CH in the development of t-AML/MDS and demonstrate the importance of understanding specific treatment <-> mutation interactions to inform choices of therapeutic interventions in patients with primary cancers. Disclosures Vassiliou: Celgene: Research Funding; KYMAB: Consultancy, Equity Ownership.

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Expression of JAK2V617I Does Not Result in Overt Myeloproliferative Disorder, but Results in Cytokine Hypersensitivity

Blood ◽

10.1182/blood.v124.21.3218.3218 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3218-3218

Author(s):

Stefan Brooks ◽

Samuel B Luty ◽

Hew Yeng Lai ◽

Lacey R Royer ◽

Sarah J Morse ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Spleen Weight ◽

Hematopoietic Stem ◽

Gm Csf ◽

Empty Vector ◽

Increased Risk ◽

Germline Expression

Abstract BACKGROUND: A germline JAK2V617I mutation has recently been identified in a family with hereditary thrombocytosis (Mead et al, NEJM 2012). Like acquired MPN, family members with JAK2V617I have thrombocytosis and megakaryocytic hyperplasia in the marrow with increased risk of thrombosis. But unlike acquired MPN, individuals with this germline mutation do not develop a fibrotic bone marrow, splenomegaly, or transform to acute leukemia. Why germline JAK2V617I recapitulates some aspects but not others of the MPN phenotype in humans is unclear. To delineate the differences between JAK2V617F and JAK2V617I we compared the phenotype of mice with hematopoietic cells expressing JAK2V617F or JAK2V617I. METHODS AND RESULTS: Lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with retrovirus expressing JAK2V617F, JAK2V617I, or empty MSCV-IRES-GFP (MIG) vector. As expected, mice transplanted with JAK2V617F-expressing cells developed erythrocytosis and leukocytosis, whereas mice transplanted with JAK2V617I-expressing cells had peripheral blood counts similar to empty vector mice. Humans with germline JAK2V617I do not display constitutive activation of the kinase, but they do demonstrate cytokine hyper-responsiveness as evidenced by increased phosphorylation of STATs at low concentrations of ligand. We compared phosphorylated STAT5 in peripheral blood of mice transplanted with JAK2V617I, JAK2V617F, and MIG empty vector following stimulation with increasing concentrations of GM-CSF. At all concentrations of GM-CSF tested JAK2V617I and JAK2V617F-expressing cells had exaggerated phosphorylation of STAT5 as compared to MIG empty vector mice. We also measured phospho-STAT3 and STAT5 in unstimulated bone marrow and spleen from each mouse at time of euthanasia, there was no difference between JAK2V617I and MIG empty vector mice. JAK2V617F mice did demonstrate phosphorylation of STAT3 and STAT5 in the absence of GM-CSF, confirming the ability of JAK2V617F but not JAK2V617I to constitutively activate downstream signaling pathways. Next, to evaluate for histologic evidence of MPN and assess spleen size, all mice were euthanized at 120 days post-transplant. JAK2V617F mice had splenomegaly as expected, spleens from JAK2V617I mice appeared larger than empty vector mice, but spleen weight was not statistically different (p>0.05). While JAKV617I mice had increased cellularity of their marrow with increased numbers of megakaryocytes as compared to empty vector mice, this was not nearly to the extent of JAK2V617F mice. Mild fibrosis was seen in JAK2V617I mice, JAK2V617F mice had severe reticulin fibrosis in the marrow as expected. In the spleen architecture was preserved in the JAK2V617I mice, whereas in the JAK2V617F mice splenic architecture was disrupted by invasion of myeloid cells including megakaryocytes. To identify whether JAK2V617I affects the frequency of stem and progenitor cells or expands mature myeloid lineage cells we measured the frequency of hematopoietic stem cells, myeloid progenitors, and mature myeloid populations in JAK2V617F, JAK2V617I, and MIG empty vector. The bone marrow of JAK2V617I mice contained an increased percentage of GMP and MEP populations as compared to both the MIG empty vector and the JAK2V617F mice. Mature granulocyte (Gr-1+CD11b+) and erythroid (Ter119+) populations were expanded in the bone marrow and spleen of JAK2V617F but not JAK2V617I mice. CONCLUSIONS: These data demonstrate that the JAK2V617I mouse model recapitulates the effect of germline expression of JAK2V617I seen in humans: it results in cytokine hyper-responsiveness without the ability to constitutively activate downstream signals in the absence of ligand. Why JAK2V617F is so exquisitely conserved in acquired MPN is still unknown. Disclosures No relevant conflicts of interest to declare.

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The Role of Gja1 Gap Junctions in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v108.11.1392.1392 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1392-1392

Author(s):

Nicole M. Anderson ◽

Ralph Zirngibi ◽

Frieda Chen ◽

Celeste Owen ◽

Lia Zitano ◽

...

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Gap Junctions ◽

Longitudinal Analysis ◽

Peripheral Blood ◽

Peripheral Blood Cell ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Junction Protein ◽

The Impact

Abstract The interplay between the stroma and hematopoietic progenitors within the bone marrow niche is critical for the homeostatic regulation of both mesenchymal and blood lineages. Gap junctions play an important role in the communication between hematopoietic supportive cells of the fetal liver and bone marrow stroma. Targeted deletion of the gap junction protein Gja1 (connexin 43) demonstrated a requirement of Gja1 for placental and cardiac function leading to neonatal lethality. Surprisingly, Gja-1 heterozygous animals demonstrated no steady state hematopoietic defects, suggesting that either Gja-1 is not critical for hematopoietic stromal cell communication or that Gja-1 hemizygosity does not functionally alter gap junctions. We have identified an N-ethyl-N-nitrosourea (ENU)-induced strain (Gja1JRT) with a dominant negative mutation in Gja1 which phenocopies the human autosomal dominant disorder oculodentodigital dysplasia (ODDD). ODDD is characterized by a variety of developmental abnormalities including syndactyly, enamel hypoplasia, craniofacial abnormalities and cardiac dysfunction. In addition to these phenotypes, Gja-1JRT mice exhibit decreased bone mass and mechanical strength as well as alterations in hematopoietic progenitor frequencies, abnormalities not previously reported in human ODDD patients (Development132: 4375–4386, 2005). We are currently performing a longitudinal analysis of 2, 4, 8 and 12 month old Gja-1JRT animals, examining hematopoietic including osteoclastic and mesenchymal/osteoblast progenitors to evaluate the dynamic interplay between these two tissues. To date, we have observed alterations in hematopoietic parameters including increased peripheral blood cell number and increased myeloid colony number at 2 months of age. At 4 months of age, Gja-1JRT mice demonstrated a significant decrease in overall clonogenic (CFU-C) cells, while some peripheral blood cell parameters remained elevated. Despite the increases in differentiated myeloid cells and their precursors at 2 months of age, analysis of hematopoietic-derived bone resorbing osteoclasts showed a reduction in osteoclasts at 2 months of age but not at later time points. Analysis of the mesenchymal compartment of the bone marrow demonstrated increased total mesenchymal progenitors (CFU-F) and osteoprogenitors (CFU-O) at 8 and 12 months of age. Thus, alterations in hematopoietic and osteoclastic cell frequencies preceded alterations in the bone marrow mesenchymal compartment in the Gja-1JRT mice. In addition to a complete longitudinal analysis, we will report on the alterations in trabecular bone, which create the niche microenvironment, and the impact of these changes on the hematopoietic stem cell frequency.

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Impact of Tet2 Deficiency, and of TET2 Mutations in Clonal Hematopoiesis, on Neutrophil/Granulocyte Immune Function

Blood ◽

10.1182/blood-2021-147095 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2159-2159

Author(s):

Elina K Cook ◽

Michael Luo ◽

Jeffrey Mewburn ◽

Kimberly J Dunham-Snary ◽

Charles Hindmarch ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Immune Function ◽

Mouse Serum ◽

Rna Seq ◽

Granulocytic Differentiation ◽

Multiple Testing Correction ◽

Hematopoietic Stem ◽

Clonal Hematopoiesis ◽

Increased Risk

Abstract BACKGROUND: Neutrophils, the most abundant leukocytes and granulocytes, are important regulators of cardiovascular, inflammatory and infectious diseases, yet their role in the pathophysiology of clonal hematopoiesis of indeterminate potential (CHIP) has not been adequately addressed. The effects of inactivating CHIP-driver mutations in the epigenetic regulator TET2 in neutrophils especially, are broadly unknown. HYPOTHESIS: Tet2 inactivation in murine neutrophils, and TET2 mutations in CHIP in humans (CHIP TET2), perturb granulocyte immune effector functions. METHODS: Neutrophils were obtained (EasySep™, StemCell) from the bone marrow of 2- to 4-months-old, sex-matched, control Tet2 f/f;Vav1-icre - (Tet2 f/f) and hematopoietic knockout Tet2 f/f;Vav1-icre + (Tet2 -/-) mice. Neutrophils were cultured (RPMI+10% mouse serum/FBS) and: i) stained with Mitotracker Deep Red/Nuc Blue, co-cultured and imaged (Leica SP8-X) for 30min with GFP-labeled Staphylococcus aureus (10:1 ratio) and analyzed in FIJI; ii) cultured for 3h with vehicle or 10μg/mL of S. aureus lipotechoic acid (LTA). RNA-Seq was generated (Illumina QuantSeq 3' mRNA, single-end 75bp read lengths, 5 million reads/sample), trimmed, aligned to GRCm39 using STAR. CHIP participant DNA and RNA were sequenced previously from whole blood (Cook et al., Bld Adv 2019; Cook et al., ASH 2018, with a 48-gene panel on Ion Proton, and ribo-depleted bulk RNA on Illumina, respectively). New CHIP TET2 vs. no CHIP, and murine RNA-Seq analyses were carried out in DESeq2. Human serum granule protein levels were quantified by ELISA (VersaMax). Mann-Whitney U tests were carried out in Prism. P<0.05 was considered statistically significant, and Benjamini-Hochberg multiple testing correction was applied as needed. RESULTS: Tet2 -/- mice had 1.34-fold more bone marrow CD11b +Ly6G + neutrophils than control Tet2 f/f mice (p=0.03), consistent with myeloid expansion. Compared to Tet2 f/f, Tet2 -/- neutrophils phagocytosed fewer S. aureus (Fig1A) and moved more slowly (Fig1B). Preliminary data suggest that Tet2 -/- neutrophil extracellular trap (NET) formation in response to S. aureus was also impaired, showing fewer and less extensive NETs (Fig1C). LTA-stimulated gene expression profiles were similar between Tet2 -/- and Tet2 f/f, suggesting pre-existing differences at baseline. Unexpectedly, the most significant GO term enrichment related to upregulated viral response pathways, including interferon-stimulated genes, (e.g. Ifitm1). The cause is unknown, but this is reminiscent of the constitutive interferon response seen in myelodysplastic syndrome (MDS) patients and TET2-mutant hematopoietic stem cells, where epigenetic dysregulation of endogenous retrotransposable elements leads to a viral mimicry response. Tet2 -/- neutrophils also overexpressed Asprv1, a regulator of inflammation ostensibly acquired from a retrotransposon. Interestingly, Ccdc80, which has been linked to Tet2 and Jak2 functions, was most significantly downregulated in Tet2 -/-, along with the Pnpla1 lipid phosphatase. Finally, Tesc, a promoter of granulocytic differentiation, was upregulated in Tet2 -/-, and there were perturbations of genes encoding neutrophil granule contents. Similarly, human RNA-Seq revealed that several leukocyte (de)granulation-related genes (e.g. lactoferrin LTF, myeloperoxidase MPO) were upregulated in CHIP TET2 subjects to those without CHIP, and these corresponded with higher LTF and MPO serum titers in an expanded cohort (Fig1D,E). Finally, there were striking decreases of gene expression associated with cytotoxic (T/NK) human lymphocytes (i.e. GZMM, TRGV8, etc.). Neutrophil, lymphocyte and monocyte counts were not significantly different between the groups. CONCLUSIONS: Tet2-deficient murine neutrophils have compromised immune function, possibly due to differences in pre-stimulus state. TET2-mutation carrying neutrophils in CHIP may exhibit similar abnormalities, as has been previously noted in neutrophils isolated from MDS patients. Indeed, CHIP is now known to associate with increased risk of bacterial and viral infections, and infection risk has also previously been noted for MDS. People with CHIP have elevated peripheral blood serum MPO and LTF levels, suggesting a difference in leukocyte granule biology, likely related to neutrophils. These data aid in understanding how CHIP alters immunity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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The Impact of Age and Gender on Hematopoietic Stem Cells and Immune Contexture of the Bone Marrow Microenvironment

Cells Tissues Organs ◽

10.1159/000510774 ◽

2020 ◽

pp. 1-6

Author(s):

Rebar N. Mohammed

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Absolute Number ◽

Cell Number ◽

Hematopoietic Stem ◽

The Absolute ◽

And Gender ◽

The Impact

Hematopoietic stem cells (HSCs) are a rare population of cells that reside mainly in the bone marrow and are capable of generating and fulfilling the entire hematopoietic system upon differentiation. Thirty-six healthy donors, attending the HSCT center to donate their bone marrow, were categorized according to their age into child (0–12 years), adolescence (13–18 years), and adult (19–59 years) groups, and gender into male and female groups. Then, the absolute number of HSCs and mature immune cells in their harvested bone marrow was investigated. Here, we report that the absolute cell number can vary considerably based on the age of the healthy donor, and the number of both HSCs and immune cells declines with advancing age. The gender of the donor (male or female) did not have any impact on the number of the HSCs and immune cells in the bone marrow. In conclusion, since the number of HSCs plays a pivotal role in the clinical outcome of allogeneic HSC transplantations, identifying a younger donor regardless the gender is critical.

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Elevating body temperature enhances hematopoiesis and neutrophil recovery after total body irradiation in an IL-1–, IL-17–, and G-CSF–dependent manner

Blood ◽

10.1182/blood-2012-02-409805 ◽

2012 ◽

Vol 120 (13) ◽

pp. 2600-2609 ◽

Cited By ~ 19

Author(s):

Maegan L. Capitano ◽

Michael J. Nemeth ◽

Thomas A. Mace ◽

Christi Salisbury-Ruf ◽

Brahm H. Segal ◽

...

Keyword(s):

Heat Treatment ◽

Bone Marrow ◽

Body Temperature ◽

Total Body Irradiation ◽

Dependent Manner ◽

Intestinal Tissue ◽

Hematopoietic Stem ◽

Neutrophil Recovery ◽

Total Body ◽

The Impact

Abstract Neutropenia is a common side effect of cytotoxic chemotherapy and radiation, increasing the risk of infection in these patients. Here we examined the impact of body temperature on neutrophil recovery in the blood and bone marrow after total body irradiation (TBI). Mice were exposed to either 3 or 6 Gy TBI followed by a mild heat treatment that temporarily raised core body temperature to approximately 39.5°C. Neutrophil recovery was then compared with control mice that received either TBI alone heat treatment alone. Mice that received both TBI and heat treatment exhibited a significant increase in the rate of neutrophil recovery in the blood and an increase in the number of marrow hematopoietic stem cells and neutrophil progenitors compared with that seen in mice that received either TBI or heat alone. The combination treatment also increased G-CSF concentrations in the serum, bone marrow, and intestinal tissue and IL-17, IL-1β, and IL-1α concentrations in the intestinal tissue after TBI. Neutralizing G-CSF or inhibiting IL-17 or IL-1 signaling significantly blocked the thermally mediated increase in neutrophil numbers. These findings suggest that a physiologically relevant increase in body temperature can accelerate recovery from neutropenia after TBI through a G-CSF–, IL-17–, and IL-1–dependent mechanism.

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