Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

CSF3R mutated myeloid neoplasms: Beyond chronic neutrophilic leukemia.

CSF3R activating mutation is a genetic hallmark of chronic neutrophilic leukemia (CNL), and is also present in a subset of atypical chronic myeloid leukemia (aCML), but infrequent in other myeloid neoplasms. However, the occurrence of CSF3R mutations in various myeloid neoplasms is not well studied. Here we evaluate the spectrum of CSF3R mutations and the clinicopathologic features of CSF3R mutated myeloid neoplasms. We retrospectively identified CSF3R mutations in a variety of myeloid neoplasms: two CNL, three atypical chronic myeloid leukemia (aCML), nine acute myeloid leukemia (AML), one chronic myelomonocytic leukemia, and one myeloproliferative neoplasm. The prototypic T618I mutation was found in 50% of cases: CNL (2/2), aCML (2/3) and AML (4/9). We observed a new recurrent CSF3R mutation Q776* in 25% of cases, and a potential-germline mutation in a 20-year-old patient. Co-occurring mutations were often in epigenetic modifier and spliceosome. IDH/RUNX1 and tumor suppressor mutations were frequent in AML but absent in CNL/aCML. All CNL/aCML patients succumbed within 2-years of diagnosis. We demonstrate that CSF3R mutations are not restricted to CNL. CNL and aCML show similar clinicopathologic and molecular features, suggesting that CNL may be best classified as myelodysplastic/myeloproliferative neoplasm rather than myeloproliferative neoplasm.

Myeloproliferative neoplasms in the adolescent and young adult population: A comprehensive review of the literature.

Myeloproliferative neoplasms (MPN) are characterized by a clonal proliferation of myeloid lineage cells within the bone marrow. The classical BCR-ABL negative MPNs are comprised of polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. Historically, the majority of MPNs are diagnosed in adults older than 60 years of age; however, in recent years, there has been recognition of MPNs in the adolescent and young adult (AYA) population. AYAs with MPN, typically defined as between the ages of 15 and 39 years old, may comprise up to 20% of patients diagnosed with MPN. They demonstrate unique patterns of driver mutations and thrombotic events and remain at risk for progression to more aggressive disease states. Given the likely long length of time they will live with their disease, there is a significant unmet need in identifying well-tolerated and effective treatment options for these patients, particularly with the advent of disease modification. In this review, we provide a comprehensive overview of the clinical features, disease course and management of AYA patients with MPN and, in doing so, highlight key characteristics that distinguish them from their older counterparts.

Assessment of Total Antioxidant Capacity, 8-Hydroxy-2'-deoxy-guanosine, the Genetic Landscape, and Their Associations in BCR::ABL-1-Negative Chronic and Blast Phase Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders defined by an excessive production of functionally mature and terminally differentiated myeloid cells. MPNs can transform into secondary acute myeloid leukemia (sAML/blast phase MPN) and are linked to alterations in the redox balance, i.e., elevated concentrations of reactive oxygen species and markers of oxidative stress (OS), and changes in antioxidant systems. We evaluated OS in 117 chronic phase MPNs and 21 sAML cases versus controls by measuring total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxy-guanosine (8-OHdG) concentrations. TAC was higher in MPNs than controls (p = 0.03), particularly in ET (p = 0.04) and PMF (p = 0.01). MPL W515L-positive MPNs had higher TAC than controls (p = 0.002) and triple-negative MPNs (p = 0.01). PMF patients who had treatment expressed lower TAC than therapy-free subjects (p = 0.03). 8-OHdG concentrations were similar between controls and MPNs, controls and sAML, and MPNs and sAML. We noted associations between TAC and MPNs (OR = 1.82; p = 0.05), i.e., ET (OR = 2.36; p = 0.03) and PMF (OR = 2.11; p = 0.03), but not sAML. 8-OHdG concentrations were not associated with MPNs (OR = 1.73; p = 0.62) or sAML (OR = 1.89; p = 0.49). In conclusion, we detected redox imbalances in MPNs based on disease subtype, driver mutations, and treatment history.

Molecular Pathology of Myeloid Neoplasms: Molecular Pattern Recognition.

Despite the apparent complexity of the molecular genetic underpinnings of myeloid neoplasms, most myeloid mutational profiles can be understood within a simple framework. Somatic mutations accumulate in hematopoietic stem cells with aging and toxic insults, termed clonal hematopoiesis. These "old stem cells" mutations, predominantly in the epigenetic and RNA spliceosome pathways, act as "founding" driver mutations leading to a clonal myeloid neoplasm when sufficient in number and clone size. Subsequent mutations can create the genetic flavor of the myeloid neoplasm ("backseat" drivers) due to their enrichment in certain entities or act as progression events ("aggressive" drivers) during clonal evolution.

Increased CXCL10 (IP-10) is associated with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models.

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Immunophenotype of myeloid granulocytes in Chinese patients with BCR::ABL1-negative myeloproliferative neoplasms.

Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Trisomy 8 Defines a Distinct Subtype of Myeloproliferative Neoplasms Driven by the MYC-Alarmin Axis.

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.

Clonal hematopoiesis-derived therapy-related myeloid neoplasms after autologous hematopoietic stem cell transplant for lymphoid and non-lymphoid disorders.

Therapy-related myeloid neoplasms (tMN) are complications of cytotoxic therapies. Risk of tMN is high in recipients of autologous hematopoietic stem cell transplantation (aHSCT). Acquisition of genomic mutations represents a key pathogenic driver but the origins, timing and dynamics, particularly in the context of preexisting or emergent clonal hematopoiesis (CH), have not been sufficiently clarified. We studied a cohort of 1507 patients undergoing aHSCT and a cohort of 263 patients who developed tMN without aHSCT to determine clinico-molecular features unique to post-aHSCT tMN. We show that tMN occurs in up to 2.3% of patients at median of 2.6 years post-AHSCT. Age ≥ 60 years, male sex, radiotherapy, high treatment burden ( ≥ 3 lines of chemotherapy), and graft cellularity increased the risk of tMN. Time to evolution and overall survival were shorter in post-aHSCT tMN vs. other tMN, and the earlier group's mutational pattern was enriched in PPM1D and TP53 lesions. Preexisting CH increased the risk of adverse outcomes including post-aHSCT tMN. Particularly, antecedent lesions affecting PPM1D and TP53 predicted tMN evolution post-transplant. Notably, CH-derived tMN had worse outcomes than non CH-derived tMN. As such, screening for CH before aHSCT may inform individual patients' prognostic outcomes and influence their prospective treatment plans. Presented in part as an oral abstract at the 2022 American Society of Hematology Annual Meeting, New Orleans, LA, 2022.

Cabot rings in a cat with myeloproliferative disease.

A 6-year-old spayed female Scottish Fold cat presented with lethargy and anorexia. A complete blood cell count indicated severe anemia and mild thrombocytopenia. Examination of peripheral blood smears revealed marked changes in the erythroid lineage, including the presence of basophilic stippling and Howell-Jolly bodies as well as an increase in nucleated erythrocytes, polychromatophils, ovalocytes, and schistocytes. Additionally, some erythrocytes contained a ring or figure-eight shaped structure known as a Cabot ring, which were especially observed in polychromatophilic erythrocytes. Hemolytic diseases (Mycoplasma infection and IMHA) were diagnostically excluded, and the cat was treated through prednisolone administration, whole blood transfusion, and administration of vitamins (K2 and B12); however, the anemia progressively worsened. Cabot rings were observed until Day 22 and subsequently disappeared as the number of nucleated RBCs increased, and the erythrocyte lineage shifted to immature population. On Day 42, peripheral blood examination revealed further left shifting and appearance of many rubriblasts. The patient died at home on Day 43. Necropsy revealed neoplastic cells infiltrating the bone marrow and other organs, which were immunopositive to CD71 which is an erythroid lineage marker. In humans, Cabot rings have been observed in megaloblastic anemia, lead poisoning, myelodysplastic syndrome, and myelofibrosis; further, they are thought to be related to stressed bone marrow and dyserythropoiesis. This is the first case report of a cat with Cabot rings, which are suggestive of defects in erythroid lineage production.

CCN2/CTGF expression does not correlate with fibrosis in myeloproliferative neoplasms, consistent with noncanonical TGF-ß signaling driving myelofibrosis.

The classical BCR::ABL1-negative myeloproliferative neoplasms (MPN) form a group of bone marrow (BM) diseases with the potential to progress to acute myeloid leukemia or develop marrow fibrosis and subsequent BM failure. The mechanism by which BM fibrosis develops and the factors that drive stromal activation and fibrosis are not well understood. Cellular Communication Network 2 (CCN2), also known as CTGF (Connective Tissue Growth Factor), is a profibrotic matricellular protein functioning as an important driver and biomarker of fibrosis in a wide range of diseases outside the marrow. CCN2 can promote fibrosis directly or by acting as a factor downstream of TGF-ß, the latter already known to contribute to myelofibrosis in MPN.To study the possible involvement of CCN2 in BM fibrosis in MPN, we assessed CCN2 protein expression by immunohistochemistry in 75 BM biopsies (55 × MPN and 20 × normal controls). We found variable expression of CCN2 in megakaryocytes with significant overexpression in a subgroup of 7 (13%) MPN cases; 4 of them (3 × essential thrombocytemia and 1 × prefibrotic primary myelofibrosis) showed no fibrosis (MF-0), 2 (1 × post-polycythemic myelofibrosis and 1 × primary myelofibrosis) showed moderate fibrosis (MF-2), and 1 (primary myelofibrosis) severe fibrosis (MF-3). Remarkably, CCN2 expression did not correlate with fibrosis or other disease parameters such as platelet count or thrombovascular events, neither in this subgroup nor in the whole study group. This suggests that in BM of MPN patients other, CCN2-independent pathways (such as noncanonical TGF-ß signaling) may be more important for the development of fibrosis.

Gadd45g insufficiency drives the pathogenesis of myeloproliferative neoplasms.

Despite the identification of driver mutations leading to the initiation of myeloproliferative neoplasms (MPNs), the molecular pathogenesis of MPNs remains incompletely understood. Here, we demonstrate that growth arrest and DNA damage inducible gamma (GADD45g) is expressed at significantly lower levels in patients with MPNs, and JAK2V617F mutation and histone deacetylation contribute to its reduced expression. Downregulation of GADD45g plays a tumor-promoting role in human MPN cells. Gadd45g insufficiency in the murine hematopoietic system alone leads to significantly enhanced growth and self-renewal capacity of myeloid-biased hematopoietic stem cells, and the development of phenotypes resembling MPNs. Mechanistically, the pathogenic role of GADD45g insufficiency is mediated through a cascade of activations of RAC2, PAK1 and PI3K-AKT signaling pathways. These data characterize GADD45g deficiency as a novel pathogenic factor in MPNs.

The role of leukocytes in myeloproliferative neoplasm thromboinflammation.

Classic myeloproliferative neoplasms lacking the Philadelphia chromosome are stem cell disorders characterized by the proliferation of myeloid cells in the bone marrow and increased counts of peripheral blood cells. The occurrence of thrombotic events is a common complication in myeloproliferative neoplasms. The heightened levels of cytokines play a substantial role in the morbidity and mortality of these patients, establishing a persistent proinflammatory condition that culminates in thrombosis. The etiology of thrombosis remains intricate and multifaceted, involving blood cells and endothelial dysfunction, the inflammatory state, and the coagulation cascade, leading to hypercoagulability. Leukocytes play a pivotal role in the thromboinflammatory process of myeloproliferative neoplasms by releasing various proinflammatory and prothrombotic factors as well as interacting with other cells, which contributes to the amplification of the clotting cascade and subsequent thrombosis. The correlation between increased leukocyte counts and thrombotic risk has been established. However, there is a need for an accurate biomarker to assess leukocyte activation. Lastly, tailored treatments to address the thrombotic risk in myeloproliferative neoplasms are needed. Therefore, this review aims to summarize the potential mechanisms of leukocyte involvement in myeloproliferative neoplasm thromboinflammation, propose potential biomarkers for leukocyte activation, and discuss promising treatment options for controlling myeloproliferative neoplasm thromboinflammation.

In vivo ablation of NF-κB cascade effectors alleviates disease burden in myeloproliferative neoplasms.

ABSTRACT: Hyperactivation of the NF-κB cascade propagates oncogenic signaling and proinflammation, which together augments disease burden in myeloproliferative neoplasms (MPNs). Here, we systematically ablate NF-κB signaling effectors to identify core dependencies using a series of primary samples and syngeneic and patient-derived xenograft (PDX) mouse models. Conditional knockout of Rela attenuated Jak2V617F- and MPLW515L-driven onset of polycythemia vera and myelofibrosis disease hallmarks, respectively. In PDXs, RELA knockout diminished leukemic engraftment and bone marrow fibrosis while extending survival. Knockout of upstream effector Myd88 also alleviated disease burden; conversely, perturbation of negative regulator miR-146a microRNA induced earlier lethality and exacerbated disease. Perturbation of NF-κB effectors further skewed the abundance and distribution of hematopoietic multipotent progenitors. Finally, pharmacological targeting of interleukin-1 receptor-associated kinase 4 (IRAK4) with inhibitor CA-4948 suppressed disease burden and inflammatory cytokines specifically in MPN without inducing toxicity in nondiseased models. These findings highlight vulnerabilities in MPN that are exploitable with emerging therapeutic approaches.

Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F-positive myeloproliferative neoplasms.

ABSTRACT: Pegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F-positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α-treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F-positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F-positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.