Extramedullary Hematopoiesis Research Articles

Survivors of polymicrobial sepsis are refractory to G-CSF-induced emergency myelopoiesis and hematopoietic stem and progenitor cell mobilization

Sepsis survivors exhibit immune dysfunction, hematological changes, and increased risk of infection. The long-term impacts of sepsis on hematopoiesis were analyzed using a surgical model of murine sepsis, resulting in 50% survival. During acute disease, phenotypic hematopoietic stem and progenitor cells (HSPCs) were reduced in the bone marrow (BM), concomitant with increased myeloid colony-forming units and extramedullary hematopoiesis. Upon recovery, BM HSPCs were increased and exhibited normal function in the context of transplantation. To evaluate hematopoietic responses in sepsis survivors, we treated recovered sham and cecal ligation and puncture mice with a mobilizing regimen of granulocyte colony-stimulating factor (G-CSF) at day 20 post-surgery. Sepsis survivors failed to undergo emergency myelopoiesis and HSPC mobilization in response to G-CSF administration. G-CSF is produced in response to acute infection and injury to expedite the production of innate immune cells; therefore, our findings contribute to a new understanding of how sepsis predisposes to subsequent infection.

Unveiling Extramedullary Hematopoiesis: A Case Report Highlighting the Causes, Symptoms, and Management Strategies

Extramedullary hematopoiesis (EMH) serves as a compensatory mechanism in chronic hemolytic anemias, such as thalassemia, and can result in spinal cord compression. This case report highlights a 36-year-old woman with transfusion-dependent β-thalassemia (TDT) who presented with lower extremity motor deficiency, pelvic paresthesia, and bladder dysfunction. The patient had a history of lower back pain, bilateral lower limb weakness, and demonstrated poor compliance with iron chelation therapy. MRI findings indicated spinal cord compression attributable to extramedullary hematopoiesis. Due to the infeasibility of surgical intervention, the patient underwent hypertransfusion and iron chelation therapy. While neurological symptoms improved, urinary retention persisted. The patient continues to receive iron chelation treatment and undergo transfusions. Managing extramedullary hematopoiesis in thalassemia necessitates an individualized treatment approach.

Impact of genotype on multi-organ iron and complications in patients with non-transfusion-dependent β-thalassemia intermedia.

We evaluated the impact of the genotype on clinical and hematochemical features, hepatic and cardiac iron levels, and endocrine, hepatic, and cardiovascular complications in non-transfusion-dependent (NTD) β-thalassemia intermedia (TI) patients. Sixty patients (39.09 ± 11.11years, 29 females) consecutively enrolled in the Myocardial Iron Overload in Thalassemia project underwent Magnetic Resonance Imaging to quantify iron overload, biventricular function parameters, and atrial areas and to detect replacement myocardial fibrosis. Three groups of patients were identified: homozygous β+ (N = 18), heterozygous β0β+ (N = 22), and homozygous β0 (N = 20). The groups were homogeneous for sex, age, splenectomy, hematochemical parameters, chelation therapy, and iron levels. The homozygous β° genotype was associated with significantly higher biventricular end-diastolic and end-systolic volume indexes and bi-atrial area indexes. No difference was detected in biventricular ejection fractions or myocardial fibrosis. Extramedullary hematopoiesis and leg ulcers were significantly more frequent in the homozygous β° group compared to the homozygous β+ group. No association was detected between genotype and liver cirrhosis, hypogonadism, hypothyroidism, osteoporosis, heart failure, arrhythmias, and pulmonary hypertension. Heart remodelling related to a high cardiac output state cardiomyopathy, extramedullary hematopoiesis, and leg ulcers were more pronounced in patients with the homozygous β° genotype compared to the other genotypes analyzed. The knowledge of the genotype can assist in the clinical management of NTD β-TI patients.

Abstract 5281: 5-Fluorouracil decreases MDSC and the development of osteolytic bone metastases from breast cancer in mice

Abstract Bone metastases are a highly debilitating complication in more than 70% of patients with advanced breast and prostate cancer. Currently approved treatments fail to cure bone metastases or increase patient survival. Although immunotherapies can cause a durable response in some patients, it seems that their efficacy is limited in bone metastases, which could be due to a cold metastatic microenvironment. Our aim was to identify immunosuppressive factors in bone metastases and target them using a drug-repositioning type approach. We used a syngeneic model in which 4T1 triple-negative breast cancer cells derived from a Balb/C mouse are inoculated in the left cardiac ventricle of Balb/C mice to cause osteolytic bone metastases. Using flow cytometry, we found that the bone marrow of mice with 4T1 bone metastases is enriched in large cells with a high SSC index that could be myeloid-derived suppressor cells. We confirmed that the inoculation of 4T1 cells increased the amount of CD11b+ Ly6G+ Ly6Clo ROShi and CD11b+ Ly6G- Ly6Chi NOhi cells. Both cell types were isolated from bone metastases and prevented the proliferation of CD8+ T cells ex vivo, confirming they were polymorphonuclear- (PMN-) and monocytic-MDSCs (M-MDSCs), respectively. In addition, both types could differentiate into osteoclasts that cause bone loss. Reviewing the current literature, we found that FDA-approved sildenafil (a PDE-5 inhibitor), zoledronic acid (a bone resorption inhibitor), and 5-fluorouracil (5-FU, a chemotherapeutic agent) can decrease MDSCs in other cancer models. However, sildenafil (20mg/kg, i.p., every 2 days) and zoledronic acid (25µg/kg, s.c., 3x/week) did not decrease the amount of MDSCs in 4T1 bone metastases. 5-FU (i.p., 4 inoculations) at 50mg/kg prevented the 4T1-induced splenomegaly and the increase of PMN- and M-MDSCs but seemed to decrease the number of T cells in bone metastases. Thus, we tested the efficacy of a lower number of inoculations. A single inoculation of 5-FU was not efficient, while 2 or 3 inoculations of 5-FU (50mg/kg) prevented the splenomegaly and the histological damage due to extramedullary hematopoiesis in the spleen. In 4T1 bone metastases, there was a decrease of PMN-MDSCs and an increase of the number of CD4+ and CD8+ T cells. On radiographs, 2 or 3 inoculations of 5-FU (50mg/kg) significantly decreased the area of the osteolytic lesions caused by 4T1 cells (-57% and -60%, respectively, compared to placebo, P<0.005). In conclusion, our results show that functional PMN- and M-MDSCs can increase and accumulate in the microenvironment of breast cancer bone metastases and that an optimized treatment with 5-FU can prevent this expansion of MDSC, increasing T cell infiltration in bone metastases and decrease the associated bone lesions. Treatment with 5-FU could then contribute to turning bone metastases from a cold to a hot microenvironment and increase the efficacy of immunotherapies in patients. Citation Format: Pierrick G.J. Fournier, Paloma Almeida Luna, Danna Arellano, Samanta Jiménez, Felipe Olvera, Patricia Juárez. 5-Fluorouracil decreases MDSC and the development of osteolytic bone metastases from breast cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5281.

Safety of Repeated Administration of Xenogeneic Human Apoptotic State (Allocetra-OTS) in Sprague Dawley Rats.

Apoptotic cells possess immunomodulatory effects that can be utilized to treat imbalanced immune conditions. Information on the preclinical safety of such treatment is sparse. In this study, the safety of apoptotic cells (Allocetra-OTS) was assessed in a GLP toxicological study on Sprague Dawley rats. Three doses of Allocetra-OTS or vehicle were administered intravenously (IV) for 3 consecutive days. Animals in the main study were sacrificed on day 4, while animals from the recovery groups were kept for 14 or 28 days. Allocetra-OTS was well tolerated, and no adverse effects were observed in terms of body weight, clinical signs, food consumption, or ophthalmologic observation. Thus, the No Observed Adverse Effect Level (NOAEL) dose was determined as the highest dose administered. An observed elevation in immune cells was suspected to be due to Allocetra-OTS, similarly to other clinical chemistry parameters; however, it was resolved in the recovery phases. Splenomegaly and dose-related extramedullary hematopoiesis (EMH) in the red pulp were observed, with no adverse events, and were considered to be a normal and expected reaction following the IV administration of cell-based therapies. In conclusion, under the conditions of this study, Allocetra-OTS was concluded to be safe, further supporting its potential candidacy for clinical studies.

Radioprotective effectiveness of a novel delta-tocotrienol prodrug on mouse hematopoietic system against 60Co gamma-ray irradiation through inducing granulocyte-colony stimulating factor production

Considering the increasing risk of nuclear attacks worldwide, the development of develop potent and safe radioprotective agents for nuclear emergencies is urgently needed. γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have demonstrated a potent radioprotective effect by inducing the production of granulocyte-colony stimulating factor (G-CSF) in vivo. However, their application is limited because of their low bioavailability. The utilization of ester prodrugs can be an effective strategy for modifying the pharmacokinetic properties of drug molecules. In this study, we initially confirmed that DT3 exhibited the most significant potential for inducing G-CSF effects among eight natural vitamin E homologs. Consequently, we designed and synthesized a series of DT3 ester and ether derivatives, leading to improved radioprotective effects. The metabolic study conducted in vitro and in vivo has identified DT3 succinate 5b as a prodrug of DT3 with an approximately seven-fold higher bioavailability compared to DT3 alone. And DT3 ether derivative 8a were relatively stable and approximately 4 times more bioavailable than DT3 prototype. Furthermore, 5b exhibited superior ability to mitigate radiation-induced pancytopenia, enhance the recovery of bone marrow hematopoietic stem and progenitor cells, and promote splenic extramedullary hematopoiesis in sublethal irradiated mice. Similarly, 8a shown potential radiation protection, but its radiation protection is less than DT3. Based on these findings, we identified 5b as a DT3 prodrug, and providing an attractive candidate for further drug development.

Enhanced medullary and extramedullary granulopoiesis sustain the inflammatory response in lupus nephritis

ObjectivesIn SLE, deregulation of haematopoiesis is characterised by inflammatory priming and myeloid skewing of haematopoietic stem and progenitor cells (HSPCs). We sought to investigate the role of extramedullary haematopoiesis (EMH)...

Multiple myeloma in dogs: Use of the cell block technique as a new diagnostic tool.

The diagnosis of multiple myeloma (MM) in dogs may be challenging and complex. The cell blocks are a diagnostic technique that allows the characterization of neoplastic cells and, therefore, might help in the diagnosis of atypical MM. The objective of the present work is to describe three clinical cases in which the cell blocks and immunohistochemistry contributed to the definitive diagnosis of canine MM. Three dogs, one female and two males, with different clinical signs, were presented for consultation with anemia, hyperproteinemia with monoclonal gammopathy, and the presence of plasmacytosis in the bone marrow. Cytologic analysis of the spleen was performed in two dogs and was suggestive of the presence of lymphocytes or plasma cells of a neoplastic nature in one of the cases and plasma cell hyperplasia associated with extramedullary hematopoiesis in the other. Given the hypotheses of lymphoid neoplasms with a plasma cell phenotype, cell blocks from aspiration punctures were performed for immunohistochemical analysis with anti-CD3, CD20, CD79αcy, PAX5, and MUM1 antibodies. The results revealed positive staining for MUM1 in 80% of the cells in the spleen cell block and for CD20 and MUM1 in 70% of the cells in the bone marrow cell blocks, with negative staining for the other antibodies. The immunophenotyping results allowed the diagnosis of MM in the three cases and excluded other lymphoid neoplasms. This work reinforces the importance of using cell blocks in the diagnosis of neoplasms by demonstrating their potential to aid the diagnosis of MM.

Vhl deletion in Dmp1-expressing cells alters MEP metabolism and promotes stress erythropoiesis

IntroductionErythropoiesis occurs in a specialized niche in the bone marrow and is regulated in part by oxygen-sensing signaling pathways. VHL is an E3 ubiquitin ligase that regulates the degradation of hypoxia-inducible factor (HIF) proteins under normoxic conditions. In hypoxia, VHL is inactivated resulting in enhancement of HIF signaling. Methods and resultsTo investigate the effects of HIF-activation in bone cells on erythropoiesis, we performed longitudinal analyses of conditional Vhl knockout mice (Dmp1-Cre;Vhlfl/fl; VhlcKO) mice, in which Vhl is deleted primarily in osteocytes but is retained in hematopoietic cells. VhlcKO mice display elevated red blood cell counts and erythropoietin levels starting at 10 weeks of age, resembling polycythemia. Using flow cytometry, we observed increased frequency of CD71loTER119hiFSClo orthochromatophilic erythroblasts and reticulocytes in 10- and 24-week-old VhlcKO bone marrow. Features of extramedullary hematopoiesis were observed in the spleen, including red pulp hyperplasia, the presence of megakaryocytes, and increased frequency of basophilic and polychromatophilic erythroblasts and of mature stress erythroid progenitors. To investigate the mechanisms that drive the accelerated erythropoiesis in the bone marrow, we enumerated myeloid progenitors and observed higher frequencies of megakaryocyte-erythroid progenitors (MEPs) at 6 and 10 weeks of age, but MEP frequency was similar to controls at 24 weeks. Despite this normalization in MEP frequencies, bulk RNA-Seq of MEPs at this age showed upregulation of Epas1 (Hif2a), consistent with a response to hypoxia, as well as genes involved in erythrocyte development. Surprisingly, genes involved in the response to glucose were also upregulated in VhlcKO MEPs. DiscussionOur studies implicate that HIF-driven alterations in skeletal homeostasis drive changes to the bone marrow microenvironment that influence erythroid cell development and MEP metabolism.

The crosstalk between lung cancer and the bone marrow niche fuels emergency myelopoiesis.

Modest response rates to immunotherapy observed in advanced lung cancer patients underscore the need to identify reliable biomarkers and targets, enhancing both treatment decision-making and efficacy. Factors such as PD-L1 expression, tumor mutation burden, and a 'hot' tumor microenvironment with heightened effector T cell infiltration have consistently been associated with positive responses. In contrast, the predictive role of the abundantly present tumor-infiltrating myeloid cell (TIMs) fraction remains somewhat uncertain, partly explained by their towering variety in terms of ontogeny, phenotype, location, and function. Nevertheless, numerous preclinical and clinical studies established a clear link between lung cancer progression and alterations in intra- and extramedullary hematopoiesis, leading to emergency myelopoiesis at the expense of megakaryocyte/erythroid and lymphoid differentiation. These observations affirm that a continuous crosstalk between solid cancers such as lung cancer and the bone marrow niche (BMN) must take place. However, the BMN, encompassing hematopoietic stem and progenitor cells, differentiated immune and stromal cells, remains inadequately explored in solid cancer patients. Subsequently, no clear consensus has been reached on the exact breadth of tumor installed hematopoiesis perturbing cues nor their predictive power for immunotherapy. As the current era of single-cell omics is reshaping our understanding of the hematopoietic process and the subcluster landscape of lung TIMs, we aim to present an updated overview of the hierarchical differentiation process of TIMs within the BMN of solid cancer bearing subjects. Our comprehensive overview underscores that lung cancer should be regarded as a systemic disease in which the cues governing the lung tumor-BMN crosstalk might bolster the definition of new biomarkers and druggable targets, potentially mitigating the high attrition rate of leading immunotherapies for NSCLC.

Beta-Thalassemia Major and Myocardial Iron Overload: A Longitudinal Study with Magnetic Resonance Imaging.

Patients with β-thalassemia major depend on lifelong transfusion, resulting in tissue iron overload. This longitudinal retrospective observational study aims to assess myocardial and liver iron overload using magnetic resonance imaging (MRI) and investigate the lag between myocardial and liver iron unloading in β-thalassemia patients undergoing chelation therapy. Beta-thalassemia major patients with at least two MRI studies between 2016 and 2020 were enrolled. Myocardial and liver iron overload were defined as T2 ∗ less than 20 and 2.1, respectively. Outcomes included mortality, myocardial and liver T2 ∗ changes, and systolic dysfunction assessed by cardiac MRI. Fifty-five patients with a mean age of 24.62 ± 7.94 years, a mean follow-up duration of 24.3 ± 12.9 months, and a mean ferritin level of 1475.75 ± 771.12 ng/mL were enrolled. All of the abovementioned patients only took deferoxamine as the iron-chelating medication. Mortality occurred in three patients (5.5%) during follow-up. Liver T2 ∗ significantly increased (p value <0.05), while myocardial T2 ∗ showed a nonsignificant increase. Iron unloading of the myocardium was not significantly different from that of the liver and did not result in a significant lag (56% vs. 44%; p value = 0.419). Baseline myocardial T2 ∗ correlated with extramedullary hematopoiesis, weekly number of deferoxamine injections (p value <0.01), timing between the transfusions, and serum ferritin (p value <0.05). Liver T2 ∗ reduced during deferoxamine chelation therapy, while myocardial T2 ∗ remained unchanged. No significant lag was observed between myocardial and liver iron unloading. Further studies are required to elucidate these findings.

The ontogenesis and heterogeneity of basophils.

Basophils are the rarest leukocytes, but they have essential roles in protection against helminths, allergic disorders, autoimmune diseases, and some cancers. For years, the clinical significance of basophils has been neglected because of the lack of proper experimental tools to study them. The development of basophil-specific antibodies and animal models, along with genomic advances like single-cell transcriptomics, has greatly enhanced our understanding of basophil biology. Recent discoveries regarding basophils prompted us to write this review, emphasizing the basophil developmental pathway. In it, we chronologically examine the steps of basophil development in various species, which reveals the apparent advent of basophils predating IgE and basophil's IgE-independent regulatory role in primitive vertebrates. Then, we cover studies of basophil development in adult bone marrow, and compare those of murine and human basophils, introducing newly identified basophil progenitors and mature basophil subsets, as well as the transcription factors that regulate the transitions between them. Last, we discuss the heterogeneity of tissue-resident basophils, which may develop through extramedullary hematopoiesis. We expect that this review will contribute to a deeper understanding of basophil biology from the intricate aspects of basophil development and differentiation, offering valuable insights for both researchers and clinicians.

Title: The ameliorative role of Camellia sinensis extract against spleenʼs extra-medullary hematopoiesis in acute myeloproliferative disorder model in rats: Histopathological study

Title: The ameliorative role of Camellia sinensis extract against spleenʼs extra-medullary hematopoiesis in acute myeloproliferative disorder model in rats: Histopathological study

Extramedullary hematopoiesis in ribs and severe pulmonary hypertension disease following intermediate beta-thalassemia: a case report

BackgroundThalassemia is a type of congenital hemoglobinopathy that falls into the category of hemolytic anemias. Extramedullary hematopoiesis is a complication of this disease, which is a mechanism to compensate for chronic anemia in these patients, and imaging is the best diagnostic method.Case reportIn this report, a 36-year-old Caucasian female patient with intermediate beta thalassemia is presented who, at the time of referral, complained of exacerbated shortness of breath. Imaging showed diffuse expansion masses with soft tissue components in the ribs of both hemithoraxes, leading to the diagnosis of extramedullary hematopoiesis.ConclusionExtramedullary hematopoiesis in the ribs is an uncommon finding in patients with thalassemia and is a sign of the severity of the disease and a poor prognostic factor that might be preventable if blood transfusion begins at younger ages.

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.

Ghosal Hematodiaphyseal Dysplasia: Implications of Late Diagnosis and Unusual Presentation in Adults

Ghosal Hematodiaphyseal Dysplasia (GHDD) is a rare autosomal recessive disorder characterized by bone modeling failure, resulting in thickening of bone cortices with predominant diaphyseal involvement and responsive anemia to steroids. Late diagnosis of GHDD in adulthood can lead to significant morbidity and suboptimal response to steroids. Moreover, GHDD often mimics more common hematological disorders like myelodysplastic syndrome and myelofibrosis, leading to treatments such as transfusions, splenectomy, and hematopoietic stem cell transplantation. Early diagnosis is critical to enable the patient to receive the appropriate treatment. We present a case series of three Indian patients from different families with genetically proven GHDD. Despite experiencing symptoms from early childhood, all patients were diagnosed in adulthood. None of them were born out of a consanguineous marriage . Two of them, aged 43 and 56, are the oldest reported GHDD patients to date. They initiated steroid treatment in their 5th and 6th decades of life, respectively. Unlike previously reported cases, these two patients required additional drugs like Thalidomide along with steroids, and their response to treatment was not as rapid. The first patient, a 43-year-old male, had a history of anemia since childhood and became transfusion dependent after 15 years of age. He developed bicytopenia and later pancytopenia with hypocellular bone marrow , along with splenohepatomegaly and extramedullary hematopoiesis in the paravertebral and presacral areas, a novel finding in GHDD cases. Whole exome sequencing revealed a missense mutation in the TBXA2 gene. The second patient, a 56-year-old male, had a history of anemia since childhood and underwent splenectomy at 46 due to massive splenomegaly and transfusion dependency. However, his transfusion dependency persisted, and bone marrow biopsy suggested myelofibrosis. However he was negativefor myeloid mutations including JAK2. Subsequently, genetic testing revealed a homozygous missense mutation in the TBXAS1 gene. He also developed paraplegia and bladder incontinence, attributed to extramedullary hematopoiesis, which improved with steroid treatment The third patient a 23 year old man with history of anemia, lower limb tenderness and ecchymosis at 14 months of age. Evaluation then had shown hyperostosis of diaphysis of long bones of lower limbs with sparing of metaphysis and bicytopenia. Was on regular transfusions till 9 years. Re-evaluation with bone marrow studies at 9 years revealed a varying cellular marrow with dysplastic megakaryocytes and diagnosed as pediatric myelodysplatic syndrome ,He was on steroids till 13 years and he did not require further transfusions.He stopped steroids for 1 year following which there was a fall in Hb levels and recurrence of pain in both lower limbs with inability to squat. In view of history of steroid responsive anemia and thickening of diaphysis in X-rays GHDD was suspected and confirmed by genetic testing. He was restarted on steroids and there was a good response to steroids within 4 week with normalisation of hemoglobin levels . Late diagnosis of GHDD in adulthood can lead to severe complications and suboptimal treatment response. These cases highlight the importance of early diagnosis to prevent unnecessary interventions and improve patient outcomes. Unusual presentations, like extramedullary hematopoiesis, can further complicate the diagnostic process. Clinicians should maintain a high index of suspicion for GHDD in cases of refractory anemia or myelofibrosis-like features, especially in adults with an earlier history of anemia. Early recognition and appropriate management are crucial to mitigate morbidity and improve patients' quality of life

MYC-Alarmin Axis As a Novel Oncogenic Driver in a Subgroup of Triple Negative Myeloproliferative Neoplasms

Primary Myelofibrosis (PMF) is an aggressive myeloproliferative neoplasm (MPN) characterized by constitutional symptoms, cytopenias, splenomegaly, extramedullary hematopoiesis, bone marrow (BM) fibrosis, and a propensity to transform to acute myeloid leukemia (AML). Despite advances in understanding the underlying genetic abnormalities in MPN and the development of JAK2 inhibitors to treat MF, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (TN-MF) cases that lack mutations in the JAK2 kinase pathway and who have inferior outcomes. To identify potential oncogenic drivers in TN-MF, we performed cytogenetic analyses and targeted exome sequencing of 98 genes commonly mutated in myeloid malignancies in 584 MF patients identified from the Total Cancer Care database at Moffitt Cancer Center. Notably, there were no significant differences in the somatic mutation profiles (other than JAK2 activating mutations) between TN vs. JAK2/CALR/MPL mutant MF patients. However, trisomy of chromosome 8 occurs more frequently in TN-MF vs. other subtypes (26.7% vs. 6.2%, p=0.0001). To identify oncogenic drivers in trisomy 8+ TN-MF, we performed scRNA-seq analysis from 18,651 cells ( n=15,850 from 3 normal donors and n=2,801 from a trisomy 8+ TN-MF patient) and identified a total of 1,260 genes that are differentially regulated in hematopoietic stem cells (HSCs) of trisomy 8+ TN-MF compared to normal donors. Among these, 131 genes were located on chromosome 8 and MYC was one of the top 5 genes that are significantly upregulated in trisomy 8+ HSCs. Additional immunohistochemistry staining demonstrated that MYC protein levels in BM cells were significantly higher in trisomy 8+ TN-MF patients vs. trisomy 8- patients. To assess whether MYC might be a driver of MPN, we established an Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC transgenic mouse model that inducibly overexpresses MYC in HSCs following treatment of polyinosinic-polycytidylic acid (pIpC). Forced MYC expression induced profound anemia, monocytosis, megakaryocytic atypia, splenomegaly, increased BM collagen deposition/fibrosis, extramedullary hematopoiesis in spleen and liver, and significantly reduced OS vs. Mx1-Cre +/- wild type controls (median OS 258 days vs. not reached (NR), p&amp;lt;0.0001). MYC also promoted expansion of HSCs, myeloid progenitors, and Gr-1 +/CD11b + mature myeloid cells in BM and spleen; thus, MYC was sufficient to provoke MF-like disease independent of JAK2 pathway mutations. Subsequent scRNA-seq analysis of 25,232 cells ( n=13,552 from control and n=11,680 from a MYC transgenic mouse) revealed that S100a9 mRNA levels were increased by MYC in most major hematopoietic cell types. Supporting oncogenic roles of S100A9 in MYC-driven MF, deletion of S100a9 in Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC;S100a9 -/- mice abolished MYC-induced MF phenotypes and significantly improved OS (median OS NR vs. 225 days, p=0.0345). To test whether inhibition of MYC can suppress MF disease progression in vivo, lethally irradiated CD45.1 +/CD45.2 + WT mice were transplanted with BM cells harvested from CD45.2 + Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC mice that were treated with pIpC 20 weeks prior to transplant. MYCi975, a small molecule that inhibits MYC, reduced MYC and S100a9 protein levels in BM and spleen cells. Further, MYCi975 effectively suppressed MYC-driven MF phenotypes and disease progression, and significantly improved OS (median OS NR vs. 252 days, p=0.0109). In summary, our studies are the first to describe an oncogenic role of MYC in MF pathogenesis, where MYC provokes an alarmin-driven inflammatory circuit, and where the MYC-S100A9 axis represents a therapeutic vulnerability for TN-MF patients. Accordingly, our results provide a strong rationale for testing agents targeting MYC or S100A9 in early phase clinical trials in MPNs having increased MYC levels or activity.

Impact of Kidney Dysfunction on Overall Survival in Myeloproliferative Neoplasms: A Single-Center Retrospective Study

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell disorders that can lead to the deposition of fibrous tissue, and by a propensity toward extramedullary hematopoiesis. The progression of kidney function and frequency of chronic kidney disease (CKD) in patients with MPN is unknown, although CKD is linked to increased mortality. In our study, we aimed to investigate the relationship between kidney function as measured by glomerular filtration rate (eGFR) and MPN disease and risk of thrombosis. Moreover, we analyzing the risk factors for CKD, impact of different treatment modalities and effect of CKD on survival. Patients &amp; Method: We haveretrospectively screened 762 patients between Jan 1989 to May 2019 with MPN at Princess Margaret Cancer Centre. Diagnosis, of Polycythemia (PV), essential thrombocytosis (ET), and myelofibrosis (MF; comprising PMF, post-ET-PMF, and post-PV-PMF) was required as defined by the WHO classification. Patients with other MPNs (CNL, CEL, HES, MDS/MPN, MPN-U, Mastocytosis accelerating phase MPN and blast phase) or missing serial creatinine measurement or their CKD attributed to other causes (diabetic nephropathy, High blood pressure, polycystic kidney disease obstructive uropathy, Glomerulonephritis before MPN diagnosis and other) were excluded, resulting in study sample of 232 patients. The total cohort was subdivided according to the calculated eGFR, (ml/min/1.73m2) into eGFR1 (≥90, n=154), eGFR2 (60-89, n=12), and eGFR3 (&amp;lt;60, n=66). eGFR was collected retrospectively, because of small number of patients, we combined eGFR2 and eGFR3 in one group. Overall Survival (OS) was calculated using the Kaplan-Meier and log-rank test was used to assess impact variables of interest. Cox proportional hazards model was used to assess for prognostic factors of OS as well as to assess the joint effect of potential prognostic factors. Results: Median age for the total cohort was 58 years (range;18-88.3), 54% were male. Median follow-up duration was 86 months (range:45.5-135). Diagnosis of MPN involved ET; n=21(9%)), PV; n=32 (14%) and MF; n=179 (77%). JAK2V617F status was documented in 219 patients; 67% were positive; of 106 patients analyzed for CARL 40.6% were positive. Cardiovascular risk factors were higher in PV and MF than in ET (59.4%, 57% &amp;38%) respectively. A higher uric acid and LDH levels were found in PV and MF. A total of 19% had a history of thrombosis and rate was higher in ET (28.6%) than PV (21.9%) or MF (16.8%). Further patient's characteristics summarized in Table1. Kidney biopsy performed in 8 patients. The most prominent histological finding included focal segmental glomerulosclerosis (n=3), Ig A nephropathy (n=3), mesangial hypercellularity and sclerosis, extramedullary haematopoiesis (n=1) and lupus nephritis (n=1). The risk factors for CKD based on eGFR group are presented in (Table 2). MPN diagnosis is a significant risk factor for kidney function (p=0.0117). In MF a higher rate of eGFR 88.5% compared with PV and ET. JAK2V617 demonstrates a significant impact on abnormal eGFR (p=0.05);. IPSS and DIPSS score are a significant risk factor for kidney dysfunction (p=0.0014, and 0.0074 respectively). High uric acid levels and neutrophil counts were higher in high eGFR group (p=0.0014, and 0.0074 respectively). No association found between thrombosis and high eGFR. Hydroxyurea treatment does not have an impact on CKD, however, more patients received Ruxolitinb in abnormal eGFR (p=0.001). CKD has significant impact on OS. The 5 years OS for eGFR1 was 75% (95% CI 67%-81%), and for abnormal eGFR was 63.9% (95% CI 52%-73%), p=0004 (Fig 1). Conclusion: Higher incidence of kidney dysfunction associated in MF compared to PV and ET, there was no association between thrombosis and high eGFR as we excluded all other cause of CKD. MF with abnormal eGFR associated with high DIPSS score. MPN patients with kidney dysfunction significantly affects OS, which indicate close monitoring and prospective study is required.

Fedratinib Overcomes Ruxolitinib Resistance through Inhibition of the Interferon Signaling Pathway

Background: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by hyperactive JAK-STAT signaling, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly. Ruxolitinib (RUX) and Fedratinib are both JAK inhibitors used for the treatment of patients with intermediate or higher risk MF. While RUX is the first line treatment for patients with MF, about 50% of the patients discontinue treatment due to lack of efficacy or intolerance (Harrison et al., Ann. Hematol. 2020) after 3 years. Fedratinib is used as a second line therapy in MF based on efficacy from the JAKARTA2 study. The recently concluded FREEDOM and FREEDOM2 studies further support this. In this work, using in vitro approaches, we have explored the mechanistic basis of fedratinib's ability to overcome RUX resistance. Pharmacologically, Fedratinib binds with high affinity to the substrate-binding site and with low affinity to the ATP-binding site of JAK2(Kesarwani etal., Sci. Rep. 2015) while RUX binds only to the ATP-binding site. Previous studies on RUX resistance have shown acquired mutations driving resistance to RUX but not to Fedratinib. Here, we use RUX sensitive and resistant BaF3 (expressing human JAK2 WT or V617F) cell lines to examine the mechanism of resistance to RUX and the mechanism of efficacy of Fedratinib in RUX resistance. Methods: BaF3 cell lines overexpressing JAK2 WT or JAK2 activating mutation V617F were incrementally exposed to RUX over 3 weeks (up to IC 90) to induce resistance. Cell proliferation and phosphorylation of STAT5 in Fedratinib and RUX-treated cells were measured using celltiter glo and immunoblotting, respectively. RNAseq and ATAC-seq analyses were performed in triplicates by treating the relevant cell lines with DMSO, RUX or Fedratinib at two timepoints (6 and 15 hours). Pathway analysis was performed on RNAseq data using Metacore®. Results and Discussion: Ruxolitinib resistant BaF3 JAK2V617F cells showed minimal inhibition of proliferation (&amp;lt;10%) when treated with RUX but maintained sensitivity towards Fedratinib (IC 50 650nM vs 1552nM in RUX sensitive and resistant cells respectively) in 24-hour proliferation assays. Biochemical analysis revealed that Fedratinib inhibited JAK-STAT signaling in the RUX resistant cell line while RUX was unable to do so as measured by p-STAT5. Similar results were observed in BaF3-JAK2-WT cells as well. To further understand the mechanism of RUX-resistance and differential activity of RUX and Fedratinib, we performed RNAseq analysis on our RUX sensitive and resistant models before and after RUX and Fedratinib treatment. Myelofibrosis is associated with aberrant cytokine production and pro-inflammatory signaling that are mediated through activation of TNF-α and NF-κB signaling (Dunbar et al., Blood 2023). Consistent with this, RNAseq analysis of BaF3 JAK2 V617F cells treated with RUX or Fedratinib showed effective inhibition of these pathways by both agents as early as 6 hours. In the RUX resistant cells, while RUX did not have any significant pathway level changes, the top enriched pathways upon treatment with Fedratinib were predominantly interferon signaling pathways which includes JAK-STAT signaling suggesting that Fedratinib's efficacy in MF is not impacted by RUX resistance. To understand the mechanism of resistance, comparison of gene expression data from RUX senstive and RUX resistant cells without any drug treatment revealed minimal gene expression/pathway level differences suggesting that the mechanism of resistance could be driven by genetic (Downes et al. NPJ Precis Oncol, 2021) or epigenetic changes(Dunbar et al., Blood 2023). Ongoing analyses includes whole genome sequencing and ATAC-seq to investigate potential mutations and chromatin accessibility changes during resistance acquisition to RUX and how Fedratinib may overcome these. Conclusion: Our stable RUX resistant preclinical model demonstrates that Fedratinib is effective in overcoming RUX resistance by continuing to target key pathways in MPN biology including interferon signaling. Resistance to RUX on the other hand seems to be driven through potential genetic or epigenetic changes based on the minimal changes observed in gene expression profiles. Our ongoing analyses will reveal these potential genetic and epigenetic modifications associated with RUX resistance and explain the differential activity of RUX versus Fedratinib.

The Mechanism of Setd2 in Mesenchymal Stromal Cell in MDS-Associated Ineffective Hematopoiety

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal, hematopoietic stem progenitor cell malignant disease. Bone marrow microenvironment participates in the occurrence and development of MDS and plays a key role. Histone methyltransferase Setd2 is the only enzyme that can catalyze the methylation of mammalian histone H3K36Me3. Previous studies have confirmed that Setd2 is involved in the the transformation of MDS to leukemia, and the expression of S100A9 decreases after Setd2 knockout. This study is based on NHD13 mice with mesenchymal stem cell (MSC) targeted knockout of Setd2. Through experimental methods such as flow cytometry, peripheral blood routine, Wright staining, HE staining, and Gomori staining, it finds that Setd2 targeted knockout mice undergo faster leukemia transformation compared to NHD13 mice; Peripheral blood white blood cells increase, while hemoglobin and platelets decrease; The differentiation of hematopoietic stem progenitor cells in bone marrow is abnormal, with abnormal hematopoiesis and increased blast cells; Spleen fibrosis aggravates and megakaryocyte infiltrates; and Extramedullary hematopoiesis in liver. Through flow cytometry,β- Galactosidase staining and other experimental methods, we finds that compared with NHD13 mice, the MSC morphology of knockout mice is irregular, and the support function for hematopoietic cells is weakened. This study demonstrates that MSC targeted knockout of Setd2 has a promoting effect on the progression of MDS. We used mass spectrometry to identify proteins that are significantly upregulated in the targeted knockout mouse bone marrow supernatant compared to the NHD13 mouse bone marrow supernatant, and and select the protein Uba5 related to hematopoiesis from them. The expression of Uba5 in the bone marrow supernatant of knockout mice is significantly higher than that in the NHD13 group through WB validation. It is speculated that the hematopoietic changes in Setd2 targeted knockout mice may be related to the downstream target protein Uba5 of Setd2. This study takes the change of bone marrow microenvironment as an entry point to explore the possible mechanism of MDS ineffective hematopoiesis, hoping to provide microenvironment regulation therapy for some MDS patients with genes that hematopoietic stem cell transplantation that cannot cure(such as TP53, NRAS, KRAS), and provide new ideas for MDS treatment. There are still many limitations in this study, further clinical validation and vivo and vitro rescue experiments on downstream target genes and proteins are still needed in the future.