Thrombosis risk prediction in lymphoma patients: A multi-institutional, retrospective model development and validation study.

Venous thromboembolism (VTE) poses a significant risk to cancer patients receiving systemic therapy. The generalizability of pan-cancer models to lymphomas is limited. Currently, there are no reliable risk prediction models for thrombosis in patients with lymphoma. Our objective was to create a risk assessment model (RAM) specifically for lymphomas. We performed a retrospective cohort study to develop Fine and Gray sub-distribution hazard model for VTE and pulmonary embolism (PE)/ lower extremity deep vein thrombosis (LE-DVT) respectively in adult lymphoma patients from the Veterans Affairs national healthcare system (VA). External validations were performed at the Harris Health System (HHS) and the MD Anderson Cancer Center (MDACC). Time-dependent c-statistic and calibration curves were used to assess discrimination and fit. There were 10,313 (VA), 854 (HHS), and 1858 (MDACC) patients in the derivation and validation cohorts with diverse baseline. At 6 months, the VTE incidence was 5.8% (VA), 8.2% (HHS), and 8.8% (MDACC), respectively. The corresponding estimates for PE/LE-DVT were 3.9% (VA), 4.5% (HHS), and 3.7% (MDACC), respectively. The variables in the final RAM included lymphoma histology, body mass index, therapy type, recent hospitalization, history of VTE, history of paralysis/immobilization, and time to treatment initiation. The RAM had c-statistics of 0.68 in the derivation and 0.69 and 0.72 in the two external validation cohorts. The two models achieved a clear differentiation in risk stratification in each cohort. Our findings suggest that easy-to-implement, clinical-based model could be used to predict personalized VTE risk for lymphoma patients.

Venous Thrombosis Assay in a Mouse Model of Cancer.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Mechanisms of platelet activation in cancer-associated thrombosis: a focus on myeloproliferative neoplasms.

Platelets are anucleate blood cells that play key roles in thrombosis and hemostasis. Platelets are also effector cells in malignancy and are known to home into the microenvironment of cancers. As such, these cells provide central links between the hemostatic system, inflammation and cancer progression. Activation of platelets by cancers has been postulated to contribute to metastasis and progression of local tumor invasion. Similarly, cancer-activated platelets can increase the risk of development of both arterial and venous thrombosis; a major contributor to cancer-associated morbidity. Platelet granules secretion within the tumor environment or the plasma provide a rich source of potential biomarkers for prediction of thrombotic risk or tumor progression. In the case of myeloproliferative neoplasms (MPNs), which are characterized by clonal expansion of myeloid precursors and abnormal function and number of erythrocytes, leukocytes and platelets, patients suffer from thrombotic and hemorrhagic complications. The mechanisms driving this are likely multifactorial but remain poorly understood. Several mouse models developed to recapitulate MPN phenotype with one of the driving mutations, in JAK2 (JAK2V617F) or in calreticulin (CALR) or myeloproliferative leukemia virus oncogene receptor (MPL), have been studied for their thrombotic phenotype. Variability and discrepancies were identified within different disease models of MPN, emphasizing the complexity of increased risk of clotting and bleeding in these pathologies. Here, we review recent literature on the role of platelets in cancer-associated arterial and venous thrombosis and use MPN as case study to illustrate recent advances in experimental models of thrombosis in a malignant phenotype. We address major mechanisms of tumor-platelet communication leading to thrombosis and focus on the role of altered platelets in promoting thrombosis in MPN experimental models and patients with MPN. Recent identification of platelet-derived biomarkers of MPN-associated thrombosis is also reviewed, with potential therapeutic implications.

Epidemiology of Cancer-Associated Venous Thromboembolism in Patients With Solid and Hematologic Neoplasms in the Veterans Affairs Health Care System.

Importance: Identifying changes in epidemiologic patterns of the incidence and risk of cancer-associated thrombosis (CAT), particularly with evolving cancer-directed therapy, is essential for risk stratification. Objective: To assess the incidence of CAT over time and to determine pertinent patient-specific, cancer-specific, and treatment-specific factors associated with its risk. Design, Setting, and Participants: This longitudinal, retrospective cohort study was conducted from 2006 to 2021. Duration of follow-up was from the date of diagnosis until first venous thromboembolism (VTE) event, death, loss of follow-up (defined as a 90-day gap without clinical encounters), or administrative censoring on April 1, 2022. The study took place within the US Department of Veterans Affairs national health care system. Patients with newly diagnosed invasive solid tumors and hematologic neoplasms were included in the study. Data were analyzed from December 2022 to February 2023. Exposure: Newly diagnosed invasive solid tumors and hematologic neoplasms. Main Outcomes: Incidence of VTE was assessed using a combination of International Classification of Diseases, Ninth Revision, Clinical Modification and International Statistical Classification of Diseases, Tenth Revision, Clinical Modification and natural language processing confirmed outcomes. Cumulative incidence competing risk functions were used to estimate incidence of CAT. Multivariable Cox regression models were built to assess the association of baseline variables with CAT. Pertinent patient variables included demographics, region, rurality, area deprivation index, National Cancer Institute comorbidity index, cancer type, staging, first-line systemic treatment within 3 months (time-varying covariate), and other factors that could be associated with the risk of VTE. Results: A total of 434 203 patients (420 244 men [96.8%]; median [IQR] age, 67 [62-74] years; 7414 Asian or Pacific Islander patients [1.7%]; 20 193 Hispanic patients [4.7%]; 89 371 non-Hispanic Black patients [20.6%]; 313 157 non-Hispanic White patients [72.1%]) met the inclusion criteria. Overall incidence of CAT at 12 months was 4.5%, with yearly trends ranging stably from 4.2% to 4.7%. The risk of VTE was associated with cancer type and stage. In addition to confirming well-known risk distribution among patients with solid tumors, a higher risk of VTE was observed among patients with aggressive lymphoid neoplasms compared with patients with indolent lymphoid or myeloid hematologic neoplasms. Compared with no treatment, patients receiving first-line chemotherapy (hazard ratio [HR], 1.44; 95% CI, 1.40-1.49) and immune checkpoint inhibitors (HR, 1.49; 95% CI, 1.22-1.82) had a higher adjusted relative risk than patients receiving targeted therapy (HR, 1.21; 95% CI, 1.13-1.30) or endocrine therapy (HR, 1.20; 95% CI, 1.12-1.28). Finally, adjusted VTE risk was significantly higher among Non-Hispanic Black patients (HR, 1.23; 95% CI, 1.19-1.27) and significantly lower in Asian or Pacific Islander patients (HR, 0.84; 95% CI, 0.76-0.93) compared with Non-Hispanic White patients. Conclusions and Relevance: In this cohort study of patients with cancer, a high incidence of VTE was observed, with yearly trends that remained stable over the 16-year study period. Both novel and known factors associated with the risk of CAT were identified, providing valuable and applicable insights in this current treatment landscape.

Venous thromboembolism risk in cancer patients receiving first-line immune checkpoint inhibitor versus chemotherapy.

It remains unclear if immune checkpoint inhibitor (ICI) therapy is associated with higher rate of venous thromboembolism (VTE) compared with cytotoxic chemotherapy (chemo) in patients with comparable cancer type, staging, and comorbidities. Using the national Veterans Affairs healthcare system database from 2016 to 2021, we performed a propensity score (PS)-weighted retrospective cohort study to compare the incidence of VTE in patients with selected stage III/IV cancer receiving first-line ICI versus chemo. The PS model utilized overlap weights to balance age, sex, race, treatment year, VTE history, paralysis/immobilization, prolonged hospitalization, cancer type, staging, time between diagnosis and treatment, and National Cancer Institute comorbidity index. Weighted Cox regressions with robust standard error were used to assess the hazard ratio (HR) and 95% confidence interval (CI). We found that among comparable advanced cancers, first-line ICI (n = 1823) and first-line chemo (n = 6345) had similar rates of VTE (8.49% for ICI and 8.36% for chemo at 6 months). The weighted HR was 1.06 (95% CI 0.88-1.26) for ICI versus chemo. In a subgroup analysis restricted to lung cancers, first-line ICI/chemo (n = 828), ICI monotherapy (n = 428), and chemo monotherapy (n = 4371) had similar rates of VTE (9.60% for ICI/chemo, 10.04% for ICI, and 8.91% for chemo at 6 months). The weighted HR was 1.05 (95% CI 0.77-1.42) for ICI versus chemo, and 1.08 (95% CI 0.83-1.42) for ICI/chemo versus chemo. In conclusion, ICI as a systemic therapy has a similarly elevated risk as cytotoxic chemo for VTE occurrence in cancer patients. This finding can inform future prospective studies exploring thromboprophylaxis strategies.

Cardiovascular Disease in Myeloproliferative Neoplasms: JACC: CardioOncology State-of-the-Art Review.

Myeloproliferative neoplasms are associated with increased risk for thrombotic complications. These conditions most commonly involve somatic mutations in genes that lead to constitutive activation of the Janus-associated kinase signaling pathway (eg, Janus kinase 2, calreticulin, myeloproliferative leukemia protein). Acquired gain-of-function mutations in these genes, particularly Janus kinase 2, can cause a spectrum of disorders, ranging from clonal hematopoiesis of indeterminate potential, a recently recognized age-related promoter of cardiovascular disease, to frank hematologic malignancy. Beyond thrombosis, patients with myeloproliferative neoplasms can develop other cardiovascular conditions, including heart failure and pulmonary hypertension. The authors review the pathophysiologic mechanisms of cardiovascular complications of myeloproliferative neoplasms, which involve inflammation, prothrombotic and profibrotic factors (including transforming growth factor-beta and lysyl oxidase), and abnormal function of circulating clones of mutated leukocytes and platelets from affected individuals. Anti-inflammatory therapies may provide cardiovascular benefit in patients with myeloproliferative neoplasms, a hypothesis that requires rigorous evaluation in clinical trials.

Inhibition of Osteoblast Differentiation by JAK2V617F Megakaryocytes Derived From Male Mice With Primary Myelofibrosis.

Past studies described interactions between normal megakaryocytes, the platelet precursors, and bone cell precursors in the bone marrow. This relationship has also been studied in context of various mutations associated with increased number of megakaryocytes. The current study is the first to examine the effects of megakaryocytes from transgenic mice carrying the most common mutation that causes primary myelofibrosis (PMF) in humans (JAK2V617F) on bone cell differentiation. Organ level assessments of mice using micro-computed tomography showed decreased bone volume in JAK2V617F males, compared to matching controls. Tissue level histology revealed increased deposition of osteoid (bone matrix prior mineralization) in these mutated mice, suggesting an effect on osteoblast differentiation. Mechanistic studies using a megakaryocyte-osteoblast co-culture system, showed that both wild type or JAK2V617F megakaryocytes derived from male mice inhibited osteoblast differentiation, but JAK2V617F cells exerted a more significant inhibitory effect. A mouse mRNA osteogenesis array showed increased expression of Noggin, Chordin, Alpha-2-HS-glycoprotein, Collagen type IV alpha 1 and Collagen type XIV alpha 1 (mostly known to inhibit bone differentiation), and decreased expression of alkaline phosphatase, Vascular cell adhesion molecule 1, Sclerostin, Distal-less homeobox 5 and Collagen type III alpha 1 (associated with osteogenesis) in JAK2V617F megakaryocytes, compared to controls. This suggested that the mutation re-programs megakaryocytes to express a cluster of genes, which together could orchestrate greater suppression of osteogenesis in male mice. These findings provide mechanistic insight into the effect of JAK2V617F mutation on bone, encouraging future examination of patients with this or other PMF-inducing mutations.

Characterization of Glycoproteoforms of Integrins α2 and ß1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis.

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of ß1 integrin and enhanced adhesion activity of the α2ß1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin ß1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in ß1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin ß1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and ß1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.

Myeloproliferative disorders and their effects on bone homeostasis: the role of megakaryocytes.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of chronic hematological diseases that arise from the clonal expansion of abnormal hematopoietic stem cells, of which polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) have been extensively reviewed in the context of control of clonal expansion, fibrosis, and other phenotypes. Herein, we review current knowledge on the influence of different forms of MPN on bone health. In studies, murine models and human data have implicated various degrees of effect of different forms of MPN on bone density and on osteoblast proliferation and differentiation. Most results have shown that bone volume is generally increased in patients with PMF, whereas it is slightly decreased or not altered in patients with ET or PV, although possible differences between male and female phenotypes were not fully explored in most MPN forms. Osteosclerosis in patients with PMF is a serious complication that can lead to bone marrow failure, and the loss of bone reported in some patients with ET or PV can lead to osteoporotic fractures. Some MPN forms are associated with an increased number of megakaryocytes (MKs), and several of the MK-associated factors in MPN are known to affect bone development. We review known mechanisms involved in these processes, with a focus on the role of MKs and secreted factors. Understanding MPN-associated changes in bone health could improve early intervention and treatment of this side effect of the pathology.

Integrins and their role in megakaryocyte development and function.

Mature megakaryocytes, the platelet precursors, originate from hematopoietic stem cell progenitors, which, once committed to this lineage, undergo endomitosis leading to polyploidization. The process entails repeated rounds of DNA replication without cell division, yielding polyploid cells. Supporting the cell's developmental process and various cellular functions are integrin receptors, a conduit of communication between the extracellular environment and the cell actin cytoskeleton. Integrins are heterodimers of α and ß subunits, where different combinations of the known 18 α and 8 ß subunits confer specificity to the receptor. Integrin ligands range from extracellular matrices through soluble ligands, infectious agents, and counterreceptors, to cells. In this review, we describe the different integrins expressed on bone marrow megakaryocytes and their attributed roles in lineage development and cellular functions, including adhesion, spreading, proplatelet formation, and functional interaction with other cells. Pathologies associated with dysregulated megakaryocyte integrin expression are also reviewed.

Indoleamine 2,3-dioxygenase-1, a Novel Therapeutic Target for Post-Vascular Injury Thrombosis in CKD.

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

Bone Marrow and Adipose Tissue Adenosine Receptors Effect on Osteogenesis and Adipogenesis.

Adenosine is an extracellular signaling molecule that is particularly relevant in times of cellular stress, inflammation and metabolic disturbances when the levels of the purine increase. Adenosine acts on two G-protein-coupled stimulatory and on two G-protein-coupled inhibitory receptors, which have varying expression profiles in different tissues and conditions, and have different affinities for the endogenous ligand. Studies point to significant roles of adenosine and its receptors in metabolic disease and bone health, implicating the receptors as potential therapeutic targets. This review will highlight our current understanding of the dichotomous effects of adenosine and its receptors on adipogenesis versus osteogenesis within the bone marrow to maintain bone health, as well as its relationship to obesity. Therapeutic implications will also be reviewed.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

Matrix Mechanosensation in the Erythroid and Megakaryocytic Lineages.

The biomechanical properties of the bone marrow microenvironment emerge from a combination of interactions between various extracellular matrix (ECM) structural proteins and soluble factors. Matrix stiffness directs stem cell fate, and both bone marrow stromal and hematopoietic cells respond to biophysical cues. Within the bone marrow, the megakaryoblasts and erythroblasts are thought to originate from a common progenitor, giving rise to fully mature magakaryocytes (the platelet precursors) and erythrocytes. Erythroid and megakaryocytic progenitors sense and respond to the ECM through cell surface adhesion receptors such as integrins and mechanosensitive ion channels. While hematopoietic stem progenitor cells remain quiescent on stiffer ECM substrates, the maturation of the erythroid and megakaryocytic lineages occurs on softer ECM substrates. This review surveys the major matrix structural proteins that contribute to the overall biomechanical tone of the bone marrow, as well as key integrins and mechanosensitive ion channels identified as ECM sensors in context of megakaryocytosis or erythropoiesis.

Lysyl oxidase inhibition in primary myelofibrosis: A renewed strategy.

Primary myelofibrosis (PMF) is a type of myeloproliferative neoplasm (MPN) that portends a poor prognosis and has limited options for treatment. PMF is often driven by clonal mutations in one of three genes that regulate the JAK-STAT signaling pathway, leading to hyperactivation of this signaling pathway and over-proliferation of megakaryocytes (MKs) and their precursors. PMF presents with debilitating symptoms such as splenomegaly and weight loss. The few available treatments for PMF include a JAK2 inhibitor, ruxolitinib, which causes side effects and is not always effective. The extracellular matrix (ECM) and bone marrow (BM) microenvironment may play an important role in the pathogenesis of PMF. Lysyl oxidase (LOX), an enzyme that plays a key role in the ECM by facilitating the cross-linking of collagen and elastin fibers, has been shown to be upregulated in MKs of PMF mice and in PMF patients, suggesting its role in the progression of BM fibrosis. Recently, LOX has been identified as a potential novel therapeutic target for PMF and the development of new small molecule LOX inhibitors, PXS-LOX_1 and PXS-LOX_2, has shown some promise in slowing the progression of PMF in pre-clinical studies. Given that these inhibitors displayed an ability to target the dysregulation of the ECM via LOX inhibition, they show promise as therapeutic agents for an underappreciated aspect of PMF.

Metabolites in a mouse cancer model enhance venous thrombogenicity through the aryl hydrocarbon receptor-tissue factor axis.

Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

JAK2 V617F -Mediated Clonal Hematopoiesis Accelerates Pathological Remodeling in Murine Heart Failure.

Janus kinase 2 (valine to phenylalanine at residue 617) (JAK2 V617F ) mutations lead to myeloproliferative neoplasms associated with elevated myeloid, erythroid, and megakaryocytic cells. Alternatively these same mutations can lead to the condition of clonal hematopoiesis with no impact on blood cell counts. Here, a model of myeloid-restricted JAK2 V617F expression from lineage-negative bone marrow cells was developed and evaluated. This model displayed greater cardiac inflammation and dysfunction following permanent left anterior descending artery ligation and transverse aortic constriction. These data suggest that JAK2 V617F mutations arising in myeloid progenitor cells may contribute to cardiovascular disease by promoting the proinflammatory properties of circulating myeloid cells.

Novel lysyl oxidase inhibitors attenuate hallmarks of primary myelofibrosis in mice.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) that usually portends a poor prognosis with limited therapeutic options available. Currently, only allogeneic stem cell transplantation is curative in those who are candidates, while administration of the JAK1/2 inhibitor ruxolitinib carries a risk of worsening cytopenia. The limited therapeutic options available highlight the need for the development of novel treatments for PMF. Lysyl oxidase (LOX), an enzyme vital for collagen cross-linking and extracellular matrix stiffening, has been found to be upregulated in PMF. Herein, we evaluate two novel LOX inhibitors, PXS-LOX_1 and PXS-LOX_2, in two animal models of PMF (GATA1low and JAK2V617F-mutated mice). Specifically, PXS-LOX_1 or vehicle was given to 15- to 16-week-old GATA1low mice via intraperitoneal injection at a dose of 15 mg/kg four times a week for 9 weeks. PXS-LOX_1 was found to significantly decrease the bone marrow fibrotic burden and megakaryocyte number compared to vehicle in both male and female GATA1low mice. Given these results, PXS-LOX_1 was then tested in 15- to 17-week-old JAK2V617F-mutated mice at a dose of 30 mg/kg four times a week for 8 weeks. Again, we observed a significant decrease in bone marrow fibrotic burden. PXS-LOX_2, a LOX inhibitor with improved oral bioavailability, was next evaluated in 15- to 17-week-old JAK2V617F-mutated mice at a dose of 5 mg/kg p.o. four times a week for 8 weeks. This inhibitor also resulted in a significant decrease in bone marrow fibrosis, albeit with a more pronounced amelioration in female mice. Taking these results together, PXS-LOX_1 and PXS-LOX_2 appear to be promising new candidates for the treatment of fibrosis in PMF.