Modeling skeletal dysplasia in Hurler syndrome using patient-derived bone marrow osteoprogenitor cells.

Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet-based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.

Expansion of memory Vδ2 T cells following SARS-CoV-2 vaccination revealed by temporal single-cell transcriptomics.

γδ T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.

New pan-ALK inhibitor-resistant EML4::ALK mutations detected by liquid biopsy in lung cancer patients.

ALK and ROS1 fusions are effectively targeted by tyrosine kinase inhibitors (TKIs), however patients inevitably relapse after an initial response, often due to kinase domain mutations. We investigated circulating DNA from TKI-relapsed NSCLC patients by deep-sequencing. New EML4::ALK substitutions, L1198R, C1237Y and L1196P, were identified in the plasma of NSCLC ALK patients and characterized in a Ba/F3 cell model. Variants C1237Y and L1196P demonstrated pan-inhibitor resistance across 5 clinical and 2 investigational TKIs.

Late relapse of chronic myeloid leukemia after allogeneic bone marrow transplantation points to KANSARL (KANSL1::ARL17A) alteration: a case report with insights on the molecular landscape.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome and the consequent BCR::ABL1 oncoprotein. In the era before the introduction of tyrosine kinase inhibitors (TKIs), the only potentially curative treatment was allogeneic hematopoietic stem cell transplantation (HSCT). Here, we present the case of a patient affected by CML who experienced a relapse 20 years after allogeneic HSCT. Following relapse, the patient was treated with imatinib and bosutinib, resulting in a deep molecular response and successfully discontinued treatment. Additional analysis including whole-exome sequencing and RNA sequencing provided some insights on the molecular mechanisms of the relapse: the identification of the fusion transcript KANSL1::ARL17A (KANSARL), a cancer predisposition fusion gene, could justify a condition of genomic instability which may be associated with the onset and/or probably the late relapse of his CML.

Control-FREEC viewer: a tool for the visualization and exploration of copy number variation data.

BACKGROUND: Copy number alterations (CNAs) are genetic changes commonly found in cancer that involve different regions of the genome and impact cancer progression by affecting gene expression and genomic stability. Computational techniques can analyze copy number data obtained from high-throughput sequencing platforms, and various tools visualize and analyze CNAs in cancer genomes, providing insights into genetic mechanisms driving cancer development and progression. However, tools for visualizing copy number data in cancer research have some limitations. In fact, they can be complex to use and require expertise in bioinformatics or computational biology. While copy number data analysis and visualization provide insights into cancer biology, interpreting results can be challenging, and there may be multiple explanations for observed patterns of copy number alterations. RESULTS: We created Control-FREEC Viewer, a tool that facilitates effective visualization and exploration of copy number data. With Control-FREEC Viewer, experimental data can be easily loaded by the user. After choosing the reference genome, copy number data are displayed in whole genome or single chromosome view. Gain or loss on a specific gene can be found and visualized on each chromosome. Analysis parameters for subsequent sessions can be stored and images can be exported in raster and vector formats. CONCLUSIONS: Control-FREEC Viewer enables users to import and visualize data analyzed by the Control-FREEC tool, as well as by other tools sharing a similar tabular output, providing a comprehensive and intuitive graphical user interface for data visualization.

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.

TWIST1 Upregulation Is a Potential Target for Reversing Resistance to the CDK4/6 Inhibitor in Metastatic Luminal Breast Cancer Cells.

Cyclin-dependent kinase (CDK) 4/6 inhibitors have significantly improved progression-free survival in hormone-receptor-positive (HR+), human-epidermal-growth-factor-receptor-type-2-negative (HER2-) metastatic luminal breast cancer (mLBC). Several studies have shown that in patients with endocrine-sensitive or endocrine-resistant LBC, the addition of CDK4/6 inhibitors to endocrine therapy significantly prolongs progression-free survival. However, the percentage of patients who are unresponsive or refractory to these therapies is as high as 40%, and no reliable and reproducible biomarkers have been validated to select a priori responders or refractory patients. The selection of mutant clones in the target oncoprotein is the main cause of resistance. Other mechanisms such as oncogene amplification/overexpression or mutations in other pathways have been described in several models. In this study, we focused on palbociclib, a selective CDK4/6 inhibitor. We generated a human MCF-7 luminal breast cancer cell line that was able to survive and proliferate at different concentrations of palbociclib and also showed cross-resistance to abemaciclib. The resistant cell line was characterized via RNA sequencing and was found to strongly activate the epithelial-to-mesenchymal transition. Among the top deregulated genes, we found a dramatic downregulation of the CDK4 inhibitor CDKN2B and an upregulation of the TWIST1 transcription factor. TWIST1 was further validated as a target for the reversal of palbociclib resistance. This study provides new relevant information about the mechanisms of resistance to CDK4/6 inhibitors and suggests potential new markers for patients' follow-up care during treatment.

Neuroactive Steroid-Gut Microbiota Interaction in T2DM Diabetic Encephalopathy.

The pathological consequences of type 2 diabetes mellitus (T2DM) also involve the central nervous system; indeed, T2DM patients suffer from learning and memory disabilities with a higher risk of developing dementia. Although several factors have been proposed as possible contributors, how neuroactive steroids and the gut microbiome impact brain pathophysiology in T2DM remain unexplored. On this basis, in male Zucker diabetic fatty (ZDF) rats, we studied whether T2DM alters memory abilities using the novel object recognition test, neuroactive steroid levels by liquid chromatography-tandem mass spectrometry, hippocampal parameters using molecular assessments, and gut microbiome composition using 16S next-generation sequencing. Results obtained reveal that T2DM worsens memory abilities and that these are correlated with increased levels of corticosterone in plasma and with a decrease in allopregnanolone in the hippocampus, where neuroinflammation, oxidative stress, and mitochondrial dysfunction were reported. Interestingly, our analysis highlighted a small group of taxa strictly related to both memory impairment and neuroactive steroid levels. Overall, the data underline an interesting role for allopregnanolone and microbiota that may represent candidates for the development of therapeutic strategies.

Evolutionary signatures of human cancers revealed via genomic analysis of over 35,000 patients.

Recurring sequences of genomic alterations occurring across patients can highlight repeated evolutionary processes with significant implications for predicting cancer progression. Leveraging the ever-increasing availability of cancer omics data, here we unveil cancer's evolutionary signatures tied to distinct disease outcomes, representing "favored trajectories" of acquisition of driver mutations detected in patients with similar prognosis. We present a framework named ASCETIC (Agony-baSed Cancer EvoluTion InferenCe) to extract such signatures from sequencing experiments generated by different technologies such as bulk and single-cell sequencing data. We apply ASCETIC to (i) single-cell data from 146 myeloid malignancy patients and bulk sequencing from 366 acute myeloid leukemia patients, (ii) multi-region sequencing from 100 early-stage lung cancer patients, (iii) exome/genome data from 10,000+ Pan-Cancer Atlas samples, and (iv) targeted sequencing from 25,000+ MSK-MET metastatic patients, revealing subtype-specific single-nucleotide variant signatures associated with distinct prognostic clusters. Validations on several datasets underscore the robustness and generalizability of the extracted signatures.

Antibody Deficiency in Patients with Biallelic KARS1 Mutations.

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

Effects of blocking CD24 and CD47 'don't eat me' signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia.

Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) with a poor prognosis, at high risk of relapse after conventional treatment. MCL-associated tumour microenvironment (TME) is characterized by M2-like tumour-associated macrophages (TAMs), able to interact with cancer cells, providing tumour survival and resistance to immuno-chemotherapy. Likewise, monocyte-derived nurse-like cells (NLCs) present M2-like profile and provide proliferation signals to chronic lymphocytic leukaemia (CLL), a B-cell malignancy sharing with MCL some biological and phenotypic features. Antibodies against TAMs targeted CD47, a 'don't eat me' signal (DEMs) able to quench phagocytosis by TAMs within TME, with clinical effectiveness when combined with Rituximab in pretreated NHL. Recently, CD24 was found as valid DEMs in solid cancer. Since CD24 is expressed during B-cell differentiation, we investigated and identified consistent CD24 in MCL, CLL and primary human samples. Phagocytosis increased when M2-like macrophages were co-cultured with cancer cells, particularly in the case of paired DEMs blockade (i.e. anti-CD24 + anti-CD47) combined with Rituximab. Similarly, unstimulated CLL patients-derived NLCs provided increased phagocytosis when DEMs blockade occurred. Since high levels of CD24 were associated with worse survival in both MCL and CLL, anti-CD24-induced phagocytosis could be considered for future clinical use, particularly in association with other agents such as Rituximab.

Transcriptomic profile of TNFhigh MAIT cells is linked to B cell response following SARS-CoV-2 vaccination.

Introduction: Higher frequencies of mucosal-associated invariant T (MAIT) cells were associated with an increased adaptive response to mRNA BNT162b2 SARS-CoV-2 vaccine, however, the mechanistic insights into this relationship are unknown. In the present study, we hypothesized that the TNF response of MAIT cells supports B cell activation following SARS-CoV-2 immunization. Methods: To investigate the effects of repeated SARS-CoV-2 vaccinations on the peripheral blood mononuclear cells (PBMCs), we performed a longitudinal single cell (sc)RNA-seq and scTCR-seq analysis of SARS-CoV-2 vaccinated healthy adults with two doses of the Pfizer-BioNTech BNT162b2 mRNA vaccine. Collection of PBMCs was performed 1 day before, 3 and 17 days after prime vaccination, and 3 days and 3 months following vaccine boost. Based on scRNA/TCR-seq data related to regulatory signals induced by the vaccine, we used computational approaches for the functional pathway enrichment analysis (Reactome), dynamics of the effector cell-polarization (RNA Velocity and CellRank), and cell-cell communication (NicheNet). Results: We identified MAIT cells as an important source of TNF across circulating lymphocytes in response to repeated SARS-CoV-2 BNT162b2 vaccination. The TNFhigh signature of MAIT cells was induced by the second administration of the vaccine. Notably, the increased TNF expression was associated with MAIT cell proliferation and efficient anti-SARS-CoV-2 antibody production. Finally, by decoding the ligand-receptor interactions and incorporating intracellular signaling, we predicted TNFhigh MAIT cell interplay with different B cell subsets. In specific, predicted TNF-mediated activation was selectively directed to conventional switched memory B cells, which are deputed to high-affinity long-term memory. Discussion: Overall, our results indicate that SARS-CoV-2 BNT162b2 vaccination influences MAIT cell frequencies and their transcriptional effector profile with the potential to promote B cell activation. This research also provides a blueprint for the promising use of MAIT cells as cellular adjuvants in mRNA-based vaccines.

Risk of progression in chronic phase-chronic myeloid leukemia patients eligible for tyrosine kinase inhibitor discontinuation: Final analysis of the TFR-PRO study.

Disease progression to accelerated/blast phase (AP/BP) in patients with chronic phase chronic myeloid leukemia (CP-CML) after treatment discontinuation (TD) has never been systematically reported in clinical trials. However, recent reports of several such cases has raised concern. To estimate the risk of AP/BP among TD-eligible patients, we conducted TFR-PRO, a cohort retro-prospective study: 870 CP-CML patients eligible for TD formed a discontinuation cohort (505 patients) and a reference one (365 patients). The primary objective was the time adjusted rate (TAR) of progression in relation to TD. Secondary endpoints included the TAR of molecular relapse, that is, loss of major molecular response (MMR). With a median follow up of 5.5 years and 5188.2 person-years available, no events occurred in the TD cohort. One event of progression was registered 55 months after the end of TD, when the patient was contributing to the reference cohort. The TAR of progression was 0.019/100 person-years (95% CI [0.003-0.138]) in the overall group; 0.0 (95% CI [0-0.163]) in the discontinuation cohort; and 0.030 (95% CI [0.004-0.215]) in the reference cohort. These differences are not statistically significant. Molecular relapses occurred in 172/505 (34.1%) patients after TD, and in 64/365 (17.5%) patients in the reference cohort, p < .0001. Similar rates were observed in TD patients in first, second or third line of treatment. CML progression in patients eligible for TD is rare and not related to TD. Fears about the risk of disease progression among patients attempting TD should be dissipated.

Characterization of cancer subtypes associated with clinical outcomes by multi-omics integrative clustering.

Cancer patients show heterogeneous phenotypes and very different outcomes and responses even to common treatments, such as standard chemotherapy. This state-of-affairs has motivated the need for the comprehensive characterization of cancer phenotypes and fueled the generation of large omics datasets, comprising multiple omics data reported for the same patients, which might now allow us to start deciphering cancer heterogeneity and implement personalized therapeutic strategies. In this work, we performed the analysis of four cancer types obtained from the latest efforts by The Cancer Genome Atlas, for which seven distinct omics data were available for each patient, in addition to curated clinical outcomes. We performed a uniform pipeline for raw data preprocessing and adopted the Cancer Integration via MultIkernel LeaRning (CIMLR) integrative clustering method to extract cancer subtypes. We then systematically review the discovered clusters for the considered cancer types, highlighting novel associations between the different omics and prognosis.

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.

Myelodysplastic Syndromes/Myeloproliferative Overlap Neoplasms and Differential Diagnosis in the WHO and ICC 2022 Era: A Focused Review.

The myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) category comprises a varied group of myeloid neoplastic diseases characterized by clinical and pathologic overlapping features of both myelodysplastic and myeloproliferative neoplasms. For these reasons, these tumors are challenging in terms of diagnosis. The recent World Health Organization (WHO) 2022 classification and the International Consensus Classification (ICC) made changes in the classification of MDS/MPN compared to the previous 2016 WHO classification and improved the diagnostic criteria of these entities. The aim of this review is to describe the main entities reported in the more recent classifications, focusing on chronic myelomonocytic leukemia (CMML), MDS/MPN with neutrophilia (or atypical CML [aCML]), and MDS/MPN with SF3B1 mutation and thrombocytosis/MDS/MPN with ring sideroblasts and thrombocytosis. A particular emphasis is given to the differential diagnosis and analysis of subtle divergences and semantic differences between the WHO classification and the ICC for these entities.

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.

The chemerin/CMKLR1 axis regulates intestinal graft-versus-host disease.

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium-induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.