SBDSR126T rescues survival of sbds -/- zebrafish in a dose-dependent manner independently of Tp53.

Defects in ribosomal biogenesis profoundly affect organismal development and cellular function, and these ribosomopathies produce a variety of phenotypes. One ribosomopathy, Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, pancreatic exocrine insufficiency, and skeletal anomalies. SDS results from biallelic mutations in SBDS, which encodes a ribosome assembly factor. Some individuals express a missense mutation, SBDS R126T , along with the common K62X mutation. We reported that the sbds-null zebrafish phenocopies much of SDS. We further showed activation of Tp53-dependent pathways before the fish died during the larval stage. Here, we expressed SBDS R126T as a transgene in the sbds -/- background. We showed that one copy of the SBDS R126T transgene permitted the establishment of maternal zygotic sbds-null fish which produced defective embryos with cdkn1a up-regulation, a Tp53 target involved in cell cycle arrest. None survived beyond 3 dpf. However, two copies of the transgene resulted in normal development and lifespan. Surprisingly, neutropenia persisted. The surviving fish displayed suppression of female sex differentiation, a stress response in zebrafish. To evaluate the role of Tp53 in the pathogenesis of sbds -/- fish phenotype, we bred the fish with a DNA binding deficient allele, tp53 M214K Expression of the loss-of-function tp53 M214K did not rescue neutropenia or survival in sbds-null zebrafish. Increased expression of cdkn1a was abrogated in the tp53 M214K/M214K ;sbds -/- fish. We conclude that the amount of SBDSR126T protein is important for development, inactivation of Tp53 fails to rescue neutropenia or survival in the sbds-null background, and cdkn1a up-regulation was dependent on WT tp53 We hypothesize that additional pathways are involved in the pathophysiology of SDS.

M phase-specific interaction between SBDS and RNF2 at the mitotic spindles regulates mitotic progression.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive inherited disorder caused by biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS protein is involved in ribosome biogenesis; therefore SDS is classified as a ribosomopathy. SBDS is localized at mitotic spindles and stabilizes microtubules. Previously, we showed that SBDS interacts with ring finger protein 2 (RNF2) and is degraded through RNF2-dependent ubiquitination. In this study, we investigated when and where SBDS interacts with RNF2 and the effects of the interaction on cells. We found that SBDS co-localized with RNF2 on centrosomal microtubules in the mitotic phase (M phase), whereas SBDS and RNF2 localized to the nucleolus and nucleoplasm in the interphase, respectively. The microtubule-binding assay revealed that SBDS interacted directly with microtubules and RNF2 interacted with SBDS bound to microtubules. In addition, SBDS was ubiquitinated and degraded by RNF2 during the M phase. Moreover, RNF2 overexpression accelerated mitotic progression. These findings suggest that SBDS delays mitotic progression, and RNF2 releases cells from suppression through the ubiquitination and subsequent degradation of SBDS. The interaction between SBDS and RNF2 at mitotic spindles might be involved in mitotic progression as a novel regulatory cascade.

Apoptosis of Hematopoietic Stem Cells Contributes to Bone Marrow Suppression Following Chimeric Antigen Receptor T Cell Therapy.

Chimeric antigen receptor (CAR) T cell (CAR-T) therapy represents a revolutionary treatment for patients with relapsed/refractory hematologic malignancies. However, its use can result in significant toxicities, including cytokine release syndrome (CRS), a potentially life-threatening clinical syndrome resulting from the release of proinflammatory cytokines upon T cell activation. In addition, patients who develop CRS often experience prolonged cytopenias, and those with the most severe CRS also have the longest delays in full marrow recovery. Although an association between CRS and delayed bone marrow recovery has been established, the precise mechanism underlying this phenomenon remains unknown. This study was conducted to test our hypothesis that delayed bone marrow recovery following CAR-T therapy is caused by elevation of proinflammatory cytokines, leading to apoptosis and depletion of hematopoietic stem and progenitor cells (HSPCs). SCID-beige mice bearing intraperitoneal CD19+ Raji cell tumors were treated with injection of human CD19.28z CAR T cells. Bone marrow was then harvested for analysis by flow cytometry, and HSPCs were isolated for whole-transcriptome analysis by RNA sequencing. Complete blood counts and serum cytokine levels were measured as well. A second model was developed in which SCID-beige mice were treated with murine IFN-γ (mIFN-γ), murine IL-6 (mIL-6), or both. Bone marrow was harvested, and flow cytometry assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations. SCID-beige mice bearing intraperitoneal Raji cell tumors that were treated with CAR-T therapy developed CRS, with elevations of several proinflammatory cytokines, including profound elevation of human IFN-γ. Gene set enrichment analysis of RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. Endothelial protein C receptor (EPCR)-negative HSCs, a subset of HSCs that is poised for terminal differentiation, was found to be specifically decreased in mice that were treated with CAR T cells. Furthermore, HSPCs were found to have increased levels of apoptosis upon treatment with mIFN-γ and mIL-6, whereas short-term HSCs and multipotent progenitors exhibited increases in proliferation with mIFN-γ treatment alone. The results from this study provide evidence that the elevation of proinflammatory cytokines following CAR-T therapy impacts the bone marrow through a combined mechanism: pluripotent HSCs that are exposed to elevated levels of IFN-γ and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFN-γ. These combined effects lead to depleted stores of repopulating HSCs and ultimately cytopenias. © 2023 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Decrease in dysbaric osteonecrosis severity as a result of 45-minute oxygen pre-breathe.

Sudden decompression can result in bubble formation as the result of nitrogen gas (N2) dissolved in tissue during disabled submarine escape (DISSUB). This may cause dysbaric osteonecrosis (DON), a condition in long bones where bubbles in fatty marrow result in ischemia and necrosis. Previous research has shown that oxygen (O2) pre-breathe of two hours resulted in a reduction of DON; however, effects of shorter O2 pre-breathe remain uncertain. This study's aim was to understand the effect of shorter lengths of O2 pre-breathe. Eight adult Suffolk ewes (89.5± 11.5 kg) were exposed to 33 feet of seawater (fsw) for 24 hours. They were placed randomly into four groups and exposed to either 45, 30 or 15 minutes of O2 (91-88%) pre-breathe; the controls received none. They were then rapidly decompressed. Alizarin complexone was later injected intravenously to visualize the extent of DON in the right and left long bones (radii, tibiae, femur and humeri). The 30- and 15-minute pre-breathe groups saw the greatest deposition. There was significant decrease of variance in the 45-minute group when compared with all other treatments, suggesting that 45 minutes of O2 pre-breathe is required to effectively increase confidence in the reduction of DON. Similar confidence was not reflected in the 30-minute and 15-minute groups: 45 minutes of pre-breathe was the minimum amount needed to effectively prevent against DON in DISSUB escape at 33 fsw. However, future research is needed to determine how to calculate effective dosages of O2 pre-breathe to prevent DON in any given scenario.

Distinct genetic pathways define pre-malignant versus compensatory clonal hematopoiesis in Shwachman-Diamond syndrome.

To understand the mechanisms that mediate germline genetic leukemia predisposition, we studied the inherited ribosomopathy Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with high risk of myeloid malignancies at an early age. To define the mechanistic basis of clonal hematopoiesis in SDS, we investigate somatic mutations acquired by patients with SDS followed longitudinally. Here we report that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. We show that germline SBDS deficiency establishes a fitness constraint that drives selection of somatic clones via two distinct mechanisms with different clinical consequences. EIF6 inactivation mediates a compensatory pathway with limited leukemic potential by ameliorating the underlying SDS ribosome defect and enhancing clone fitness. TP53 mutations define a maladaptive pathway with enhanced leukemic potential by inactivating tumor suppressor checkpoints without correcting the ribosome defect. Subsequent development of leukemia was associated with acquisition of biallelic TP53 alterations. These results mechanistically link leukemia predisposition to germline genetic constraints on cellular fitness, and provide a rational framework for clinical surveillance strategies.

Allopurinol use during pediatric acute lymphoblastic leukemia maintenance therapy safely corrects skewed 6-mercaptopurine metabolism, improving inadequate myelosuppression and reducing gastrointestinal toxicity.

BACKGROUND: Inadequate myelosuppression during maintenance therapy for acute lymphoblastic leukemia (ALL) is associated with an increased risk of relapse. One mechanism is skewed metabolism of 6-mercaptopurine (6MP), a major component of maintenance therapy, which results in preferential formation of the hepatotoxic metabolite (6-methyl mercaptopurine [6MMP]) with low levels of the antileukemic metabolite, 6-thioguanine nucleotides (6TGN). Allopurinol can modify 6MP metabolism to favor 6TGN production and reduce 6MMP. METHODS: Patients in maintenance were considered for allopurinol treatment who had the following features: (a) Grade ≥3 hepatotoxicity; (b) Grade ≥2 nonhepatic gastrointestinal (GI) toxicity; or (c) persistently elevated absolute neutrophil count (ANC) despite >150% protocol dosing of oral chemotherapy. RESULTS: From 2013 to 2017, 13 ALL patients received allopurinol: nine for hepatotoxicity, five for inadequate myelosuppression, and three for nonhepatic GI toxicity (four met multiple criteria). Allopurinol was well tolerated, without significant adverse events. Allopurinol resulted in a significant decrease in the average 6MMP/6TGN ratio (mean reduction 89.1, P = .0001), with a significant increase in 6TGN (mean 550.4, P = .0008) and a significant decrease in 6MMP (mean 13 755, P = .0013). Patients with hepatotoxicity had a significant decrease in transaminase elevation after starting allopurinol (alanine transaminase [ALT] mean decrease 22.1%, P = .02), and all with nonhepatic GI toxicity had improved symptoms. Those with inadequate myelosuppression had a significant increase in the time with ANC in goal (mean increase 26.4%, P = .0004). CONCLUSIONS: Allopurinol during ALL maintenance chemotherapy is a safe, feasible, and effective intervention for those who have altered metabolism of 6MP causing toxicity or inadequate myelosuppression.

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes.

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.

Effect of nerolidol on cyclophosphamide-induced bone marrow and hematologic toxicity in Swiss albino mice.

Cyclophosphamide (CP) is one of the commonly used anticancer drugs, but its use is limited by myelotoxicity. Nerolidol (NER) is a lipophilic, bioactive sesquiterpene reported to have neuroprotective, cardioprotective, gastroprotective, and renal protective potential, but its myeloprotective potential is underexplored. This study was aimed at evaluating the myeloid-protective potential of NER in CP-induced myelotoxic mice. NER 200 and 400 mg/kg was given orally from the first to the 14th day. CP 200 mg/kg was administered intravenously on the seventh day. At the end of the study, mice were humanly killed, and blood and bone marrow were collected and stored for hematologic, biochemical and histopathologic estimations. Bone marrow analysis revealed reduced bone marrow cellularity, α-esterase activity, colony-forming unit granulocyte-macrophage (CFU-GM) levels, colony-forming unit erythroid (CFU-E) levels, and burst-forming unit-erythroid (BFU-E) levels. Hematologic findings revealed reduced peripheral blood count and granulocyte-colony stimulating factor (G-CSF) levels, whereas biochemical analysis revealed increased malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1ß levels and reduced superoxide dismutase (SOD), catalase (CAT), and IL-10 levels. Histopathologic study further strengthened our findings. Treatment with NER significantly reversed the hematotoxic and myelotoxic aberrations and retained the structural integrity of bone marrow. Findings of the current study suggest that NER is a potential therapeutic molecule that can mitigate CP-induced hematotoxic and myelotoxic manifestations. However, more detailed studies are needed to explicate the mechanism underlying its protective effect.

Hematopoietic effects of Azadirachta indica methanolic extract in cyclophosphamide mediated myelosuppressed albino rat.

Myelosuppression or bone marrow suppression is one of the most common side effects caused by anti-cancer drugs. Certain nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics and viruses like B19 virus can also cause bone marrow suppression resulting in serious consequences like leukopenia, anemia and thrombocytopenia. Currently, it is mainly treated by Filgrastim, use of which is not without side effects. Certain natural drugs can be a safer alternative to treat myelosuppression. Azadirachta indica, commonly known as Neem, is an important medicinal plant of subcontinent. Keeping in view the traditional uses of Neem, present study aims to investigate its potential role in reversing myelosuppression. Albino rats were used to determine hematopoietic activity of Neem leaves after inducing myelosuppression by cyclophosphamide given subcutaneously. Filgrastim was used as reference standard to compare the antimyelosuppressant activity of the drug. The drug was evaluated in three doses i.e. 50mg/kg, 100mg/kg and 200mg/kg body weight, while blood samples were drawn on 0, 1st, 7th, 14th and 21st day. The drug was found to be effective in reversing bone marrow suppression in all three doses based on the hematological parameters (mean WBC, RBC, platelets, Hb, Hct etc.) which improved significantly. The results suggest that the drug can be used as antimyelosuppressant after establishing its safety and identifying its active constituents with their mechanism of action.

Disease modeling of bone marrow failure syndromes using iPSC-derived hematopoietic stem progenitor cells.

The plasticity of induced pluripotent stem cells (iPSCs) with the potential to differentiate into virtually any type of cells and the feasibility of generating hematopoietic stem progenitor cells (HSPCs) from patient-derived iPSCs (iPSC-HSPCs) has many potential applications in hematology. For example, iPSC-HSPCs are being used for leukemogenesis studies and their application in various cell replacement therapies is being evaluated. The use of iPSC-HSPCs can now provide an invaluable resource for the study of diseases associated with the destruction of HSPCs, such as bone marrow failure syndromes (BMFSs). Recent studies have shown that generating iPSC-HSPCs from patients with acquired aplastic anemia and other BMFSs is not only feasible, but is also a powerful tool for understanding the pathogenesis of these disorders. In this article, we highlight recent advances in the application of iPSCs for disease modeling of BMFSs and discuss the discoveries of these studies that provide new insights in the pathophysiology of these conditions.

Prognostic value of initial bone marrow disease detection by multiparameter flow cytometry in children with neuroblastoma.

PURPOSE: Multicolor flow cytometry (MFC) is widely available, fast and has an easy-to perform approach for finding neuroblastoma (NB) cells among normal bone marrow (BM) hematopoietic cells. Aim of the study was to investigate prognostic significance of initial MFC tumor cells' detection in BM of children with NB. METHODS: 51 patients (24 boys and 27 girls) aged from 6 days to 15 years (median age 1 year 3 months) with NB were included in the study. BM samples at the time of diagnosis were obtained from 2 to 5 aspiration sites per patient. CD45(-)CD56(+)CD81(+)GD2(+)-cells were evaluated by MFC. RESULTS: NB cells were detected in BM by FC more frequently compared to conventional cytomorphology (49.0% and 29.4% patients, respectively, р = 0.043). Patients with NB cells detected in BM by MFC had significantly worse event-free survival and cumulative incidence of relapse/progression [0.24(0.08) and 0.60(0.10), respectively] compared to children with negative result of immunophenotyping [0.85(0.07) and 0.12(0.06), respectively, p < 0.001 in both cases]. BM involvement detection by MFC maintained its prognostic significance in various patients groups. In multivariate analysis, immunophenotyping proved to be an independent prognostic factor when analyzed jointly with other NB risk factors. In 42 patients BM involvement was also studied by RQ-PCR for PHOX2B and TH genes expression. Within groups of patients divided by RQ-PCR positivity, MFC-positivity retained prognostic significance. CONCLUSIONS: Thus flow cytometric BM involvement detection has very strong prognostic impact even stronger than RQ-PCR. It could be used in combination with other parameters for the treatment strategy choice in patients with NB.

Acquired Pure Red Cell Aplasia and Acquired Amegakaryocytic Thrombocytopenia Associated With Clonal Expansion of T-Cell Large Granular Lymphocytes in a Patient With Lipopolysaccharide-responsive Beige-like Anchor (LRBA) Protein Deficiency.

Acquired pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura are rare in children. Similarly, clonal expansion of T-cell large granular lymphocytes is infrequently seen in pediatrics. Lipopolysaccharide-responsive beige-like anchor (LRBA) protein deficiency is a recently described immunodeficiency syndrome that has been associated with inflammatory bowel disease and autoimmune phenomena such as Evans syndrome. Here, we describe a patient with LRBA deficiency who developed acquired pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura associated with expansion of clonal T-cell large granular lymphocytes. This has not been described in the literature previously and adds to the knowledge on the spectrum of manifestations of LRBA deficiency.

Increased Ripk1-mediated bone marrow necroptosis leads to myelodysplasia and bone marrow failure in mice.

Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate Vav CreBaxBakBid triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.

Identification of vitamin D and other bone metabolism parameters as risk factors for primary bone marrow oedema syndrome.

BACKGROUND: The aetiology and pathogenesis of primary bone marrow oedema syndrome (BMES) remain unclear. This retrospective cross-sectional study in a large cohort of patients with BMES was performed to characterise the overall skeletal status and turnover in patients with BMES, with the aim of identifying risk factors for this disease. METHODS: Patients who were diagnosed with BMES on the basis of clinical and radiological (magnetic resonance imaging) findings in our outpatient clinic were identified retrospectively. Patient history, co-existing metabolic disorders, bone metabolism parameters (serum calcium, phosphate, 25-OH-D3, bone-specific alkaline phosphatase, parathyroid hormone, and osteocalcin, and urinary deoxypyridinoline) and bone mineral density (as measured by dual-energy X-ray absorptiometry) were extracted from the medical records. Patients with secondary causes for BMES were excluded from the study. RESULTS: Of the 171 patients, 65 were identified without secondary cause for BMES. Of the 65 patients, 61.5% were female. The mean age was 49.5 ± 16.7 years, and age-related BMES prevalence showed two peaks, one in adolescence (11-20 years) and one at an older age (51-70 years). BMES predominantly affected the weight-bearing joints, namely, the ankle/foot (55.1%), knee (22.4%) and proximal femur (16.3%). Thyroid disorders and secondary hyperparathyroidism were highly prevalent (21.5 and 21.4%, respectively). On average, the cohort had elevated deoxypyridinoline levels and low 25-OH-D3 levels (19.0 ± 7.5 µg/l in patients without vitamin D supplementation). Osteopenia and osteoporosis were diagnosed in 47.4 and 17.5% of patients, respectively. CONCLUSIONS: BMES is associated with high bone turnover. Patients who are diagnosed with BMES should be screened carefully for bone metabolism disorders and their potential risk factors.

Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants.

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients' LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients' LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA-RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

Introduction to Acquired and Inherited Bone Marrow Failure.

Acquired aplastic anemia and inherited bone marrow failure syndromes both present with pancytopenia and must be distinguished because they have differences in treatment decisions and continued monitoring requirements. Advances in the genetic interrogation of patient samples have led to identification of inherited germline diseases and appreciation that patients with inherited bone marrow failure disorders may be normal in appearance with few expected clinical clues. Somatic mutations in aplastic anemia may have prognostic value. Hematopoietic stem cells from inherited marrow failure diseases can correct the proliferative defect and may develop further somatic mutations that progress to myelodysplastic syndrome or acute myeloid leukemia.

Evaluation and Management of Hematopoietic Failure in Dyskeratosis Congenita.

Dyskeratosis congenita (DC) is a rare, inherited bone marrow failure (BMF) syndrome characterized by variable manifestations and ages of onset, and predisposition to cancer. DC is one of a spectrum of diseases caused by mutations in genes regulating telomere maintenance, collectively referred to as telomere biology disorders (TBDs). Hematologic disease is common in children with DC/TBD. Timely diagnosis of underlying TBD in patients with BMF affects treatment and has been facilitated by increased awareness and availability of diagnostic tests in recent years. This article summarizes the pathophysiology, evaluation, and management of hematopoietic failure in patients with DC and other TBDs.