Laura Crisafulli

Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms associated with an increased risk of progression to secondary acute myeloid leukemia (sAML). This study investigates the genomic correlates of disease progression in MDS by profiling active genomic regulatory regions and their transcriptional impact using H3K27ac chromatin immunoprecipitation–sequencing and RNA-sequencing analysis on CD34+ bone marrow progenitors cells isolated from a prospective cohort of 86 and 357 patients, respectively. Our analysis revealed distinct patterns of genomic region activation and transcriptional regulation across different disease stages such as low-risk MDS, high-risk MDS, and sAML. Unexpectedly, unsupervised clustering revealed a subset of patients with low-risk MDS displaying regulatory and transcriptional profiles similar to those of high-risk MDS and sAML, highlighting early molecular events that may predispose patients to disease progression. This subset is characterized by PU.1 genomic occupancy in regions linked to immune and inflammatory responses, increased T-cell and natural killer cell activation, and a higher frequency of SRSF2 mutations. Clinically, patients in this group exhibit greater susceptibility to infections and cardiovascular events, along with an elevated risk of disease progression, resulting in significantly reduced overall survival. Functional studies demonstrated that PU.1 inhibition suppresses MDS cell proliferation and clonogenicity, as impaired PU.1 binding inhibits the activation of key transcriptional programs involved in disease advancement. Collectively, these findings identify epigenetic factors that predispose patients with low-risk MDS to progression to high-risk MDS and, ultimately, sAML.

PURPOSE: Tumor Protein 53 (p53) expressed from gene TP53 is a seminal tumor suppressor. We aimed to characterize mutational and nonmutational mechanisms of p53 dysfunction in myelodysplastic syndromes (MDS) and to investigate their clinical effect. PATIENTS AND METHODS: We analyzed a cohort of 6,204 patients with MDS and subsets of patients with available information on RNA sequencing of tumor cells (n = 109), high-dimensional phenotype of immune cells (n = 77), and multiomics analysis (RNA sequencing and proteomics) on single cells (n = 15). An independent validation was performed on 914 patients. RESULTS: Biallelic TP53 inactivation was a powerful driver of disease progression and identified high-risk patients, regardless of variant allele frequency. Monoallelic and biallelic inactivation represent disease stages occurring as a multihit process in MDS with TP53 mutations, thus potentially refining the optimal timing of therapeutic interventions in these patients. We identified a subset of MDS (5%) characterized by TP53 wild-type and hyperexpression of abnormal p53 protein in bone marrow progenitors that exhibit dismal outcome. These patients presented upstream p53 signaling aberrations in Pi3K cascade; RAS, WNT, and NF-KB pathways; and MDM2 gene amplification, together with a downstream dysregulation of p53 targets. MDS with p53 dysfunction displayed a distinct immune dysregulation involving myeloid-derived inflammation and impaired antigen presentation, which may be a driver of their poor prognosis and provide the groundwork for innovative immunotherapies. CONCLUSION: The identification of nonmutational p53 dysfunction in MDS may lay the foundation for a mechanistic classification of myeloid neoplasms, moving beyond a purely molecular stratification. The recognition of patients with p53 dysfunction is relevant to provide correct disease-risk assessment and interventions, as well as to refine the design of clinical trials

The hematopoietic system is sustained over time by a small pool of hematopoietic stem cells (HSCs). They reside at the apex of a complex hierarchy composed of cells with progressively more restricted lineage potential, regenerative capacity, and with different proliferation characteristics. Like other somatic stem cells, HSCs are endowed with long‐term self‐renewal and multipotent differentiation ability, to sustain the high turnover of mature cells such as erythrocytes or granulocytes, and to rapidly respond to acute peripheral stresses including bleeding, infections, or inflammation. Maintenance of both attributes over time, and of the proper balance between these opposite features, is crucial to ensure the homeostasis of the hematopoietic system. Micro‐RNAs (miRNAs) are short non‐coding RNAs that regulate gene expression posttranscriptionally upon binding to specific mRNA targets. In the past 10 years they have emerged as important players for preserving the HSC pool by acting on several biological mechanisms, such as maintenance of the quiescent state while preserving proliferation ability, prevention of apoptosis, premature differentiation, lineage skewing, excessive expansion, or retention within the BM niche. miRNA‐mediated posttranscriptional fine‐tuning of all these processes constitutes a safety mechanism to protect HSCs, by complementing the action of transcription factors and of other regulators and avoiding unwanted expansion or aplasia. The current knowledge of miRNAs function in different aspects of HSC biology, including consequences of aberrant miRNA expression, will be reviewed; yet unsolved issues will be discussed.This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development

The balance of self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells; however, the underlying molecular pathways are not completely understood, including the role of micro-RNAs. To assess micro-RNA contributions, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNAs might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNAs in the physiological stem cells-to-multi-potent progenitors transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p over-expression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiologic down-regulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

Atypical chemokine receptors (ACKRs) are regulators of leukocyte traffic, inflammation, and immunity. ACKR2 is a scavenger for most inflammatory CC chemokines and is a negative regulator of inflammation. Here we report that ACKR2 is expressed in hematopoietic precursors and downregulated during myeloid differentiation. Genetic inactivation of ACKR2 results in increased levels of inflammatory chemokine receptors and release from the bone marrow of neutrophils with increased anti-metastatic activity. In a model of NeuT-driven primary mammary carcinogenesis ACKR2 deficiency is associated with increased primary tumor growth and protection against metastasis. ACKR2 deficiency results in neutrophil-mediated protection against metastasis in mice orthotopically transplanted with 4T1 mammary carcinoma and intravenously injected with B16F10 melanoma cell lines. Thus, ACKR2 is a key regulator (checkpoint) of mouse myeloid differentiation and function and its targeting unleashes the anti-metastatic activity of neutrophils in mice.

The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.

In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl^−/− mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial. © 2012 American Society for Bone and Mineral Research.

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.