Myeloproliferative neoplasms (MPNs) are clonal disorders originated by the serial acquisition of somatic mutations in hematopoietic stem/progenitor cells. The major clinical entities are represented by polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), that are caused by driver mutations affecting JAK2, MPL or CALR. Disease progression is related to molecular and clonal evolution. PV and ET can progress to secondary myelofibrosis (sMF) but can also evolve to secondary acute myeloid leukemia (sAML). PMF is associated with the highest frequency of leukemic transformation, which represents the main cause of death. sAML is associated with a dismal prognosis and clinical features that differ from those of de novo AML. The molecular landscape distinguishes sAML from de novo AML, since the most frequent hits involve TP53, epigenetic regulators, spliceosome modulators or signal transduction genes. Single cell genomic studies provide novel and accurate information about clonal architecture and mutation acquisition order, allowing the reconstruction of clonal dynamics and molecular events that accompany leukemic transformation. In this review, we examine our current understanding of the genomic heterogeneity in MPNs and how it affects disease progression and leukemic transformation. We focus on molecular events elicited by somatic mutations acquisition and discuss the emerging findings coming from single cell studies.

Pemphigus is a life-threatening autoimmune disease. Several phenotypic variants are part of this family of bullous disorders. The disease is mainly mediated by pathogenic autoantibodies, but is also directed against two desmosomal adhesion proteins, desmoglein 1 (DSG1) and 3 (DSG3), which are expressed in the skin and mucosae. By binding to their antigens, autoantibodies induce the separation of keratinocytes, in a process known as acantholysis. The two main Pemphigus variants are Pemphigus vulgaris and foliaceus. Several models of Pemphigus have been described: in vitro, ex vivo and in vivo, passive or active mouse models. Although no model is ideal, different models display specific characteristics that are useful for testing different hypotheses regarding the initiation of Pemphigus, or to evaluate the efficacy of experimental therapies. Different disease models also allow us to evaluate the pathogenicity of specific Pemphigus autoantibodies, or to investigate the role of previously not described autoantigens. The aim of this review is to provide an overview of Pemphigus disease models, with the main focus being on active models and their potential to reproduce different disease subgroups, based on the involvement of different autoantigens.

JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells.

Background: Pemphigus is a life-threatening blistering autoimmune disease. Several forms, characterized by the presence of autoantibodies against different autoantigens, have been described. In Pemphigus Vulgaris (PV), autoantibodies target the cadherin Desmoglein 3 (DSG3), while in Pemphigus foliaceous (PF) autoantibodies target the cadherin Desmoglein 1 (DSG1). Another variant, mucocutaneous Pemphigus, is characterized by the presence of IgG against both DSG1 and DSG3. Moreover, other forms of Pemphigus characterized by the presence of autoantibodies against other autoantigens have been described. With regard to animal models, one can distinguish between passive models, where pathological IgG are transferred into neonatal mice, and active models, where B cells deriving from animals immunized against a specific autoantigen are transferred into immunodeficient mice that develop the disease. Active models recreate PV and a form of Pemphigus characterized by the presence of IgG against the cadherin Desmocollin 3 (DSC3). Further approaches allow to collect sera or B/T cells from mice immunized against a specific antigen to evaluate the mechanisms underlying the onset of the disease. Objective: To develop and characterize a new active model of Pemphigus where mice express auto antibodies against either DSG1 alone, or DSG1 and DSG3, thereby recapitulating PF and mucocutaneous Pemphigus, respectively. In addition to the existing models, with the active models reported in this work, it will be possible to recapitulate and mimic the main forms of pemphigus in adult mice, thus allowing a better understanding of the disease in the long term, including the benefit/risk ratio of new therapies. Results: The new DSG1 and the DSG1/DSG3 mixed models were developed as proposed. Immunized animals, and subsequently, animals that received splenocytes from the immunized donors produce a high concentration of circulating antibodies against the specific antigens. The severity of the disease was assessed by evaluating the PV score, evidencing that the DSG1/DSG3 mixed model exhibits the most severe symptoms among those analyzed. Alopecia, erosions, and blistering were observed in the skin of DSG1, DSG3 and DSG1/DSG3 models, while lesions in the mucosa were observed only in DSG3 and DSG1/DSG3 animals. The effectiveness of the corticosteroid Methyl-Prednisolone was evaluated in the DSG1 and DSG1/DSG3 models, that showed only partial responsiveness.

Myelofibrosis (MF) originates from the stepwise acquisition of somatic mutations in Hematopoietic Stem and Progenitor Cells (HSPCs). Alongside driver events triggering JAK-STAT pathway hyperactivation, several additional mutations, usually affecting the epigenetic machinery, contribute defining therapeutic response. Specifically, JAK-inhibition (JAKi) relieves MF symptoms but rarely eradicates the neoplastic clone. To elucidate clonal dynamics associated with JAKi, we conducted a longitudinal single-cell proteogenomic study on 6 responders and 6 non-responders MF patients. Mutational analysis revealed that the mutation acquisition order determines JAKi sensitivity. Indeed, driver-only clones are highly sensitive to JAKi, while co-mutated clones persist after treatment. JAKi response is mainly limited to the differentiated myeloid compartment, while mutant HSPCs are often maintained in JAKi-responders. Co-mutated clones may evade JAKi and outcompete other neoplastic cell populations, thus contributing to disease persistence.