First-hit SETBP1 mutations cause a myeloproliferative disorder with bone marrow fibrosis.

ABSTRACT: SETBP1 mutations are found in various clonal myeloid disorders. However, it is unclear whether they can initiate leukemia, because SETBP1 mutations typically appear as later events during oncogenesis. To answer this question, we generated a mouse model expressing mutated SETBP1 in hematopoietic tissue: this model showed profound alterations in the differentiation program of hematopoietic progenitors and developed a myeloid neoplasm with megakaryocytic dysplasia, splenomegaly, and bone marrow fibrosis, prompting us to investigate SETBP1 mutations in a cohort of 36 triple-negative primary myelofibrosis (TN-PMF) cases. We identified 2 distinct subgroups, one carrying SETBP1 mutations and the other completely devoid of somatic variants. Clinically, a striking difference in disease aggressiveness was noted, with patients with SETBP1 mutation showing a much worse clinical course. In contrast to myelodysplastic/myeloproliferative neoplasms, in which SETBP1 mutations are mostly found as a late clonal event, single-cell clonal hierarchy reconstruction in 3 patients with TN-PMF from our cohort revealed SETBP1 to be a very early event, suggesting that the phenotype of the different SETBP1+ disorders may be shaped by the opposite hierarchy of the same clonal SETBP1 variants.

Mitochondrial DNA is a target of HBV integration.

Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.

Balanced SET levels favor the correct enhancer repertoire during cell fate acquisition.

Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the oncoprotein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that a condition in which SET is accumulated, the severe Schinzel-Giedion Syndrome (SGS), is characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of SGS and possibly other the SET-related disorders in humans.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Targeting the immune microenvironment in Waldenström macroglobulinemia via halting the CD40/CD40-ligand axis.

Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Liquid Biopsy in Cancer: Focus on Lymphoproliferative Disorders.

Within the context of precision medicine, the scientific community is giving particular attention to early diagnosis and intervention, guided by non-invasive methodologies. Liquid biopsy (LBx) is a recent laboratory approach consisting of a non-invasive blood draw, which allows the detection of information about potential prognostic factors, or markers to be used for diagnostic purposes; it might also allow the clinician to establish a treatment regimen and predict a patient's response. Since the discovery of circulating tumor cells (CTCs) in the nineteenth century, the possibility of integrating LBx into clinical practice has been explored, primarily because of its safeness and easy execution: indeed, compared to solid biopsy, sampling-related risks are less of a concern, and the quickness and repeatability of the process could help confirm a prompt diagnosis or to further corroborate the existence of a metastatic spreading of the disease. LBx's usefulness has been consolidated in a narrow range of oncological settings, first of all, non-small cell lung carcinoma (NSCLC), and it is now gradually being assessed also in lymphoproliferative diseases, such as acute lymphocytic leukemia (ALL), B-cell lymphomas, and multiple myeloma. The present review aims to summarize LBx's overall characteristics (such as its advantages and flaws, collection and analysis methodologies, indications, and targets of the test), and to highlight the applications of this technique within the specific field of B-cell malignancies. The perspectives on how such a simple and convenient technique could improve hemato-oncological clinical practice are broadly encouraging, yet far from a complete integration in routine clinical settings.

Characterization of SARS-CoV-2 Mutational Signatures from 1.5+ Million Raw Sequencing Samples.

We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substitutions, considering 1,585,456 high-quality raw sequencing samples, aimed at investigating the existence and quantifying the effect of mutational processes causing mutations in SARS-CoV-2 genomes when interacting with the human host. As a result, we confirmed the presence of three well-differentiated mutational processes likely ruled by reactive oxygen species (ROS), apolipoprotein B editing complex (APOBEC), and adenosine deaminase acting on RNA (ADAR). We then evaluated the activity of these mutational processes in different continental groups, showing that some samples from Africa present a significantly higher number of substitutions, most likely due to higher APOBEC activity. We finally analyzed the activity of mutational processes across different SARS-CoV-2 variants, and we found a significantly lower number of mutations attributable to APOBEC activity in samples assigned to the Omicron variant.

ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.

De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways.

Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fail to respond to imatinib or to second-generation inhibitors and progress to blast crisis. Until now, improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis remain limited. Here we present a large parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls that reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression, with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells.