Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response.

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

A Surge of DNA Damage Links Transcriptional Reprogramming and Hematopoietic Deficit in Fanconi Anemia.

Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.

Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

ZBP1 activation triggers hematopoietic stem and progenitor cell death resulting in bone marrow failure in mice.

Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.

Characterization of novel mutations in the TEL-patch domain of the telomeric factor TPP1 associated with telomere biology disorders.

Telomeres are nucleoprotein structures that protect the chromosome ends from degradation and fusion. Telomerase is a ribonucleoprotein complex essential to maintain the length of telomeres. Germline defects that lead to short and/or dysfunctional telomeres cause telomere biology disorders (TBDs), a group of rare and heterogeneous Mendelian diseases including pulmonary fibrosis, dyskeratosis congenita, and Høyeraal-Hreidarsson syndrome. TPP1, a telomeric factor encoded by the gene ACD, recruits telomerase at telomere and stimulates its activity via its TEL-patch domain that directly interacts with TERT, the catalytic subunit of telomerase. TBDs due to TPP1 deficiency have been reported only in 11 individuals. We here report four unrelated individuals with a wide spectrum of TBD manifestations carrying either heterozygous or homozygous ACD variants consisting in the recurrent and previously described in-frame deletion of K170 (K170∆) and three novel missense mutations G179D, L184R, and E215V. Structural and functional analyses demonstrated that the four variants affect the TEL-patch domain of TPP1 and impair telomerase activity. In addition, we identified in the ACD gene several motifs associated with small deletion hotspots that could explain the recurrence of the K170∆ mutation. Finally, we detected in a subset of blood cells from one patient, a somatic TERT promoter-activating mutation that likely provides a selective advantage over non-modified cells, a phenomenon known as indirect somatic genetic rescue. Together, our results broaden the genetic and clinical spectrum of TPP1 deficiency and specify new residues in the TEL-patch domain that are crucial for length maintenance and stability of human telomeres in vivo.

Why hematopoietic stem cells fail in Fanconi anemia: Mechanisms and models.

Fanconi anemia (FA) is generally classified as a DNA repair disorder, conferring a genetic predisposition to cancer and prominent bone marrow failure (BMF) in early childhood. Corroborative human and murine studies point to a fetal origin of hematopoietic stem cell (HSC) attrition under replicative stress. Along with intriguing recent insights into non-canonical roles and domain-specific functions of FA proteins, these studies have raised the possibility of a DNA repair-independent BMF etiology. However, deeper mechanistic insight is critical as current curative options of allogeneic stem cell transplantation and emerging gene therapy have limited eligibility, carry significant side effects, and involve complex procedures restricted to resource-rich environments. To develop rational and broadly accessible therapies for FA patients, the field will need more faithful disease models that overcome the scarcity of patient samples, leverage technological advances, and adopt investigational clinical trial designs tailored for rare diseases.

CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematologic abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. OBJECTIVE: This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting with rheumatologic/immunologic symptoms or severe hematologic manifestations. METHODS: Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of mesenchymal stromal cells. RESULTS: Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and nonsevere hematologic manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematologic symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM mesenchymal stromal cells in patients with DADA2 exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. CONCLUSIONS: Our exploration into the BM landscape of patients with DADA2 sheds light on the critical hematologic dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

Shwachman-Diamond syndrome due to biallelic EFL1 variants with complex and fatal clinical course in early infancy.

Shwachman-Diamond syndrome represents a clinically and genetically heterogeneous disorder. We report on an infant with a very severe, fatal clinical course caused by biallelic EFL1 variants: c.89A>G, p.(His30Arg), and c.2599A>G, p.(Asn867Asp). Functional analysis of patient-derived B-lymphoblastoid and SV40-transformed fibroblast cell lines suggests that the compound heterozygous EFL1 variants impaired mature ribosome formation leading to compromised protein synthesis, ultimately resulting in a severe form of Shwachman-Diamond syndrome.

Revisiting the first reported case of aplastic anaemia.

The great pathologist Paul Ehrlich in Berlin is commonly credited with describing the first clear case of aplastic anaemia in 1888: a 21-year-old woman who presented with haemorrhage and signs and symptoms of severe anaemia, quickly succumbing to her illness. Ehrlich's description of this patient's background and clinical course allowed individual identification. Re-analysis of this case suggests an inherited bone marrow failure syndrome as a possible additional diagnosis.

A rare homozygous variant in TERT gene causing variable bone marrow failure, fragility fractures, rib anomalies and extremely short telomere lengths with high serum IgE.

By whole exome sequencing, we identified a homozygous c.2086 C→T (p.R696C) TERT mutation in patients who present with a spectrum of variable bone marrow failure (BMF), raccoon eyes, dystrophic nails, rib anomalies, fragility fractures (FFs), high IgE level, extremely short telomere lengths (TLs), and skewed numbers of cytotoxic T cells with B and NK cytopenia. Haploinsufficiency in the other family members resulted in short TL and osteopenia. These patients also had the lowest bone mineral density Z-score compared to other BMF-patients. Danazol/zoledronic acid improved the outcomes of BMF and FFs. This causative TERT variant has been observed in one family afflicted with dyskeratosis congenita (DC), and thus, we also define a second report and new phenotype related to the variant which should be suspected in severe cases of DC with co-existent BMF, FFs, high IgE level and rib anomalies.

Long-term assessment of haematological recovery following somatic genetic rescue in a MYSM1-deficient patient: Implications for in vivo gene therapy.

MYSM1 deficiency causes inherited bone marrow failure syndrome (IBMFS). We have previously identified an IBMFS patient with a homozygous pathogenic variant in MYSM1 who recovered from cytopenia due to spontaneous correction of one MYSM1 variant in the haematopoietic compartment, an event called somatic genetic rescue (SGR). The study of the genetic and biological aspects of the patient's haematopoietic/lymphopoietic system over a decade after SGR shows that one genetically corrected haematopoietic stem cell (HSC) can restore a healthy and stable haematopoietic system. This supports in vivo gene correction of HSCs as a promising treatment for IBMFS, including MYSM1 deficiency.

Haematological features of telomere biology disorders diagnosed in adulthood: A French nationwide study of 127 patients.

Data on haematological features of telomere biology disorders (TBD) remain scarce. We describe haematological, extra-haematological characteristics and prognosis of 127 genetically confirmed TBD patients diagnosed after the age of 15. Ninety-three index cases and 34 affected relatives were included. At diagnosis of TBD, 76.3% of index cases had haematological features, half pulmonary features and a third liver features. At diagnosis, bone marrow failure (BMF) was present in 59 (46.5%), myelodysplastic syndrome (MDS) in 22 (17.3%) and acute myeloid leukaemia (AML) in 2 (1.6%) while 13 (10.2%) developed or worsened bone marrow involvement during follow-up. At diagnosis, compared to MDS/AML patients, BMF patients were younger (median 23.1 years vs. 43.8, p = 0.007), and had a better outcome (4-year overall survival 76.3% vs. 31.8%, p < 0.001). While frequencies and burden of cytogenetical and somatic mutations increased significantly in myeloid malignancies, some abnormalities were also observed in patients with normal blood counts and BMF, notably somatic spliceosome variants. Solid cancers developed in 8.7% patients, mainly human papillomavirus-related cancers and hepatocellular carcinomas. TBD is a multiorgan progressive disease. While BMF is the main haematological disorder, high-risk myeloid malignancies are common, and are, together with age, the only factors associated with a worse outcome.

Genomic insights into inherited bone marrow failure syndromes in a Korean population.

Inherited bone marrow failure syndromes (IBMFS) pose significant diagnostic challenges due to overlapping symptoms and variable expressivity, despite evolving genomic insights. The study aimed to elucidate the genomic landscape among 130 Korean patients with IBMFS. We conducted targeted next-generation sequencing (NGS) and clinical exome sequencing (CES) across the cohort, complemented by whole genome sequencing (WGS) and chromosomal microarray (CMA) in 12 and 47 cases, respectively, with negative initial results. Notably, 50% (n = 65) of our cohort achieved a genomic diagnosis. Among these, 35 patients exhibited mutations associated with classic IBMFSs (n = 33) and the recently defined IBMFS, aplastic anaemia, mental retardation and dwarfism syndrome (AmeDS, n = 2). Classic IBMFSs were predominantly detected via targeted NGS (85%, n = 28) and CES (88%, n = 29), whereas AMeDS was exclusively identified through CES. Both CMA and WGS aided in identifying copy number variations (n = 2) and mutations in previously unexplored regions (n = 2). Additionally, 30 patients were diagnosed with other congenital diseases, encompassing 13 distinct entities including inherited thrombocytopenia (n = 12), myeloid neoplasms with germline predisposition (n = 8), congenital immune disorders (n = 7) and miscellaneous genomic conditions (n = 3). CES was particularly effective in revealing these diverse diagnoses. Our findings underscore the significance of comprehensive genomic analysis in IBMFS, highlighting the need for ongoing exploration in this complex field.

Decreased spermatogonial numbers in boys with severe haematological diseases.

This study examines spermatogonial numbers in testicular samples from 43 prepubertal patients undergoing haematopoietic stem cell transplantation (HSCT). High-dose chemotherapy and/or radiation during HSCT can impact spermatogenesis requiring fertility preservation. Results show that 49% of patients have decreased and 19% severely depleted spermatogonial pool prior to HSCT. Patients with Fanconi anaemia exhibit significantly reduced spermatogonial numbers. Patients with immunodeficiency or aplastic anaemia generally present within the normal range, while results in patients with myelodysplastic syndrome or myeloproliferative neoplasm vary. The study emphasizes the importance of assessing spermatogonial numbers in patients with severe haematological diseases for informed fertility preservation decisions.

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

Clinical utility of relative telomere length analysis in pediatric bone marrow failure.

INTRODUCTION: Telomere length related studies are limited in pediatric marrow failure cases due to difficulty in establishing population specific age related normograms. Moreover, there is paucity of data related to clinical relevance of telomere length in idiopathic aplastic anemia (IAA) and non telomere biology inherited bone marrow failure syndrome (IBMFS) cases. METHODOLOGY: Hence, in current study we investigated Relative telomere length (RTL) by RQ-PCR in 83 samples as: healthy controls (n = 44), IAA (n = 15) and IBMFS (n = 24). In addition, we performed chromosomal breakage studies and targeted NGS to screen for pathogenic variants. RESULTS & CONCLUSION: Median RTL was significantly different between control vs. IBMFS (p-0.002), IAA vs. IBMFS (p-0.0075) and DC vs. non-DC IBMFS (p-0.011) but not between control vs. IAA (p-0.46). RTL analysis had clinical utility in differentiating BMF cases as 75 % (9/12) of DC had short/very short telomeres compared to only 17 % (2/12) of non-DC IBMFS, 7 % (1/15) of IAA and 7 % (3/44) of controls (p < 0.001).

Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disorder characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3 per million population per year in the Western world, but 3 times higher in East Asia. Survival in severe aplastic anemia (SAA) has improved significantly due to advances in hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy, biologic agents, and supportive care. In SAA, HSCT from a matched sibling donor (MSD) is the first-line treatment. If a MSD is not available, options include immunosuppressive therapy (IST), matched unrelated donor, or haploidentical HSCT. The purpose of this guideline is to provide health care professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary evidence-based document prepared by a group of pediatric hematologists of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Hemato-Oncology (AIEOP) was discussed, modified and approved during a series of consensus conferences that started online during COVID 19 and continued in the following years, according to procedures previously validated by the AIEOP Board of Directors.

Outcome of first or second transplantation using unrelated umbilical cord blood without ATG conditioning regimen for pediatric bone marrow failure disorders.

BACKGROUND: Unrelated umbilical cord blood transplantation (UCBT) for bone marrow failure (BMF) disorders using conditioning regimens without Anti-Thymocyte Globulin (ATG) has been used as an alternative transplantation for emerging patients without matched-sibling donors. Experience with this transplant modality in children is limited, especially as a secondary treatment for transplant failure patients. PROCEDURE: We retrospectively reviewed 17 consecutive bone marrow failure patients who underwent unrelated umbilical cord blood transplantation in our center and received conditioning regimens of Total Body Irradiation (TBI) or Busulfan (BU) + Fludarabine (FLU) + Cyclophosphamide (CY). RESULTS: Among the 17 BMF patients, 15 patients were treated with first cord blood transplantation and another 2 with secondary cord blood transplantation because of graft failure after first haploidentical stem cell transplantation at days +38 and +82. All patients engrafted with a median donor cell chimerism of 50 % at days +7 (range, 16 %-99.95 %) and finally rose to 100 % at days +30. Median time to neutrophil engraftment was 19 days (range, 12-30) and time to platelet engraftment was 32 days (range, 18-61). Pre-engraftment syndrome (PES) was found in 16 patients (94.11 %, 16/17). Cumulative incidence of grades II to IV acute GVHD was 58.8 % (95 % CI: 32.7-84.9 %), and 17.6 % (95 % CI: 2.6-37.9 %) of patients developed chronic GVHD. The 3-year overall survival (OS) and failure-free survival (FFS) rates were 92.86 ± 6.88 %. CONCLUSION: UCBT is an effective alternative treatment for bone marrow failure pediatric patients. TBI/BU + FLU + CY regimen ensure a high engraftment rate for unrelated umbilical cord blood transplantation, which overcomes the difficulty of graft failure. Secondary salvage use of cord blood transplantation may still be useful for patients who have failed after other transplantation.

Diamond-Blackfan anemia, the archetype of ribosomopathy: How distinct is it from the other constitutional ribosomopathies?

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described in humans. DBA is a congenital hypoplastic anemia, characterized by macrocytic aregenerative anemia, manifesting by differentiation blockage between the BFU-e/CFU-e developmental erythroid progenitor stages. In 50 % of the DBA cases, various malformations are noted. Strikingly, for a hematological disease with a relative erythroid tropism, DBA is due to ribosomal haploinsufficiency in 24 different ribosomal protein (RP) genes. A few other genes have been described in DBA-like disorders, but they do not fit into the classical DBA phenotype (Sankaran et al., 2012; van Dooijeweert et al., 2022; Toki et al., 2018; Kim et al., 2017 [1-4]). Haploinsufficiency in a RP gene leads to defective ribosomal RNA (rRNA) maturation, which is a hallmark of DBA. However, the mechanistic understandings of the erythroid tropism defect in DBA are still to be fully defined. Erythroid defect in DBA has been recently been linked in a non-exclusive manner to a number of mechanisms that include: 1) a defect in translation, in particular for the GATA1 erythroid gene; 2) a deficit of HSP70, the GATA1 chaperone, and 3) free heme toxicity. In addition, p53 activation in response to ribosomal stress is involved in DBA pathophysiology. The DBA phenotype may thus result from the combined contributions of various actors, which may explain the heterogenous phenotypes observed in DBA patients, even within the same family.