Differentiation Of Hematopoietic Cells Research Articles

Investigating Racially Induced Metabolic and Genomic Signatures Mediating Inferior Survival in African Americans with Acute Myelogenous Leukemia

Background: African American (AA) patients experience worse survival outcomes compared to other patients with acute myelogenous leukemia (AML), independent of socioeconomic and molecular factors. 1 Indeed, our group previously demonstrated inferior survival in AA associated with the proxy for (1) genomic instability and (2) suggestion for inability to achieve protein damaging threshold among concurrent mutations. 2 In study expansion, we have confirmed worse overall survival (OS) for AA patients, and detected rapid attrition of AA patients, soon after diagnosis and therapy initiation. Given high incidence of cardiometabolic complications in AA patients, this prompted us to investigate the link between deranged metabolism and clinical outcomes. Additionally we explored nucleotide modifications that influence leukemogenesis in AA patients. In previous data, obesity correlated with poor outcomes for patients with acute promyelocytic leukemia (APL), but not in other AML, including in the CALGB cohort. 3-5 Interestingly, obesity is linked to 1) FTO/IRX3 pathways that block hematopoietic stem cell differentiation, and 2) oxidative stress, which may induce DNA damage favoring C>A and G>T nucleotide changes 6,7 and aberrant bone marrow microenvironment 8. Methods: We conducted retrospective analysis of 272 AML patients from Baylor TX, Georgetown DC, and Washington Hospital Center DC, including demographic and clinical variables (age, sex, race, BMI, comorbidities, smoking, treatments, and outcomes). APL patients were excluded. For patients with NGS data, mutation frequencies were annotated by ethnicity and prioritized by ELN 2022 classification. Chi-square and t-test evaluated differences in categorical and continuous variables respectively. Logistic binary regression was used to detect predictors for racial induced survival. SAS software was used. Results: Our expanded cohort has 272 AML patients, with 43 AA and 229 non-AA. AA patients showed worse OS than non-AA patients (Fig 1; 149 days vs 605 days, p=0.01). There were not any AA patients with favorable risk ELN 2022 disease status. Neither ELN 2022 risk groups nor presence of MDS-like mutations differentiated worse outcomes for AA patients, so we opted to examine the effect of obesity among AA and non-AA patients with intermediate and high risk AML (given no favorable risk AA patients in our cohort). Mean BMI for our AA patients was 28, with AA patients with BMI>28 showing significantly worse OS (Fig 1; 67 days vs 198 days, p=0.001), which was not present for non-AA patients. Metabolic derangement with high total cholesterol (p=0.02) and LDL (p=0.04) also segregated with worse OS for AAs (n=8 with cholesterol data), but not for other races. Nucleotide changes C>A and G>T characteristic of oxidative stress were overrepresented in AA patients with high BMI suggestive of clinical trend (33% vs 11%, p=0.06). Conclusion: Metabolic derangement in BMI and lipids correlated with inferior outcomes for AA patients only, supporting a unique pathobiology of highly aggressive AML that afflicts AA patients. Only AA patients demonstrated preference for C>A and G>T nucleotide changes, a hallmark of oxidative stress, also linked to obesity. These negative impacts support further mechanistic studies, and may present therapeutic strategies to improve metabolism, reduce oxidative stress, and suppress FTO/IRX3 pathways to induce myeloid differentiation. Figure 1: Inferior Survival For African American Patients Differentiated by BMI>28

Acute Inflammation Redirect Bone Marrow Hematopoietic Stem Cell Cycling and Differentiation Program By Reshaping Their Chromatin Architecture at Long-Term

Background: Acute and persistent inflammation induced by microbial infections/sepsis or by therapeutic interventions, such as whole-body irradiation and CAR-T therapy, triggers a rapid overwhelming immune response in the host. While these responses are well defined in the short-term, the long-term impact of the inflammatory injury on the hematopoietic system still needs to be unveiled. Using a murine model of severe inflammation induced by bacteria P. aeruginosa ( P. aer.) or LPS, our group has previously reported that bone marrow (BM) hematopoietic stem cells (HSC) respond to infections with an aberrant expansion associated with their transitory inability to generate the downstream myeloid progenitors. Such dysfunctions correlated with the suppression of transcription factors governing stem-cell self-renewal and myeloid differentiation. Furthermore, when transplanted into healthy mice, the “inflamed” HSC showed reduced ability to contribute to the myeloid lineage and to engraft at long-term, suggesting that they retain memory of the LPS-mediated insult and maintain long-lasting changes. Thus, we hypothesized that severe inflammation alters the HSC epigenome, reshaping their transcriptional programs and affecting cell fate decisions and differentiation. Methods: We performed time-course experiments in WT mice challenged with a single dose of LPS or PBS, as control, and investigated 1) the kinetics of hematopoietic recovery by flow-cytometric immunophenotypic characterization, including cell-cycle 2) changes in the chromatin landscape of Lin- sorted BM subpopulations identified by the expression of the CD150 and CD48 markers as Long-Term (LT-HSC), Short-Term (ST-HSC), and Multipotent Progenitors (MPP1/2 and MPP3/4) using ATAC-seq. Results: Our studies revealed that the LPS challenge resulted in a significant increase in the cell-cycle of LT-HSC, ST-HSC, MPPs, common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP), which persisted up to 3 days from the inflammatory injury. Immunophenotypic analysis showed that while HSC frequencies returned to baseline at 5 days from LPS, the abundance and cell-cycle of myeloid progenitors and mature cells remained altered. Surprisingly, at 15 days post LPS, the cell-cycle of HSC markedly decreased, and such dysregulation was maintained until our last observation at 30 days from LPS. The pool of BM HSC was severely depleted and associated with exhaustion of T-cells but increased B-cells. GMP and Gr1+Mac1+ cells were also significantly decreased in the BM whereas mature lineages were increased in the peripheral blood, exhibiting high WBC, lymphocytes, and platelet count, neutrophilia, monocytosis, basophilia, and eosinophilia. ATAC-seq analysis revealed prominent differences in the chromatin architecture of the four HSC subpopulations at steady state (PBS). In all of them, LPS induced both i) a transient reorganization of the chromatin in some loci, and ii) the formation of persistently reshaped regions. Deeper investigations into the long-term changed sites revealed that chromatic accessibility was altered in proximity of CTCF-enriched regions fundamental to mediate chromatin reorganization and drive gene expression. Moreover, LPS modified the accessibility of genomic loci involved in the maintenance of HSC stemness and quiescence as well as regulation of their cell-cycle, self-renewal, and cell fate decision. Conclusions: Taken together these results suggest that LPS-mediated acute inflammation has long-lasting effects on HSC epigenetic regulation, influencing their cell-cycle and multilineage decisions. Relevance: The association between the resulting phenotype and the persistency of epigenetically remodeled HSC loci identified here may provide potential insights for future therapeutic approaches to re-direct “inflamed” HSC to a normal differentiation program, thereby enabling an efficient innate host response. Disclosures: No relevant conflicts of interest to declare.

Avatrombopag in Combination with Immunosuppressive Therapy for Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is an uncommon but distinct variant of aplastic anemia (AA), in which an episode of seronegative acute hepatitis precedes the onset of pancytopenia. Previous studies indicated that about only 20% of HAAA patients can get their hematologic partially restored after 3 months treatment with standard immunosuppressive therapy (IST). Recent studies have shown that adding thrombopoietin receptor agonist (TPO-RA) eltrombopag to standard IST can lead to a more, faster, and better hematologic responses. Nevertheless, eltrombopag is often associated with hepatic adverse effects, i.e., elevated transaminases and bilirubin, which maybe limit its use in patients with HAAA who have just experienced severe liver damage. Avatrombopag is also a TPO-RA, which was approved for the treatment of thrombocytopenia in patients with chronic liver disease (CLD) and patients with chronic immune thrombocytopenia (ITP) by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The pre-clinical studies indicated that avatrombopag can promote the hematopoietic and megakaryocyte progenitor cells proliferation and differentiation by the dose-dependence. These in vitro effects are similar to those of other TPO-RAs, including Eltrombopag. Theoretically, on the basis of adequate IST, avatrombopag can also promote hematopoietic recovery in patients with aplastic anemia just as Eltrombopag works. If this is true, then avatrombopag should be particularly suitable for HAAA due to its favorable hepatic adverse effect profile. Nevertheless, avatrombopag added to standard IST as first-line treatment for aplastic anemia could increase the rapidity and strength of hematologic response is still unknown. Herein, we retrospectively reviewed 8 consecutive cases of patients with hepatitis-associated aplastic anemia who were treated with avatrombopag plus IST regimen. From September 2021 to December 2022, eight patients with hepatitis-associated aplastic anemia treated with avatrombopag in combination with standard IST were enrolled in this study. Among them, seven patients were diagnosed as VSAA and one SAA. The median age was 17 (9-20) years old and the median absolute neutrophil count was 0.09 (0.02-0.39) ×10 9/L. The median time from onset of hepatitis to hematopoietic failure was 2 (0.5-5.5) months, and at the time of beginning of IST all the eight patients had their liver biochemical testing returns to normal. The karyocytes were normal in all patients. One patient presented positive PNH clone at the diagnosis of HAAA, with PNH clone size of 4.7%. Avatrombopag was incorporated into day 1 of standard immunosuppressive therapy with p-ATG and cyclosporine for all the eight HAAA patients. The dose of avatrombopag was 40 mg/d at least 6 months. Dosage adjusting of avatrombopag was according to patient's platelet counts and adverse events. Encouragingly, four of the eight patients (50.0%) achieved hematologic response (HR) with two (20.0%) getting complete response (CR) at 3 months after IST and avatrombopag. Between 3 and 6 months after IST, the other two patient entered their hematologic responded. Then, six of eight (75.0%) patients got HR at 6 months, including 3 CR, 2 good partial hematologic response (GPR), and 1 partial response (PR). Unfortunately, one patient who was diagnosed as very severe HAAA, was without getting hematologic response and died at 118 days after treatment due to gastrointestinal infection. We evaluate the side effects of avatrombopag and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. No hepatic adverse events were observed in all the eight patients, that indicated avatrombopag may not increase the hepatic burden and is safe for HAAA. The median follow-up time was 371(118-665) days. Except for one patient who died within 6 months, six cases achieved hematologic response, with 4 CR and 2 GPR. There is no clonal evolution, i.e., MDS, AML, and PNH, was observed. In conclusion, avatrombopag may be used as a safe and effective TPO-RA, added to IST for the treatment of hepatitis-associated aplastic anemia, which maybe improve the efficacy and the strength of hematologic response in HAAA.

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

Bioinformatics analysis of primary biliary cholangitis key genes and molecular mechanisms

Objective: To extract the differentially expressed key genes of primary biliary cholangitis (PBC) using bioinformatics methods, so as to provide information for further study into the mechanism. Methods: The GSE119600 dataset was downloaded from the GEO database to obtain differentially expressed genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for differentially expressed genes. Protein-protein interaction (PPI) network reconstruction, Cytoscape software visualization, and core gene screening were performed. The area under the receiver operating characteristic curve (ROC AUC) was used to assess the diagnostic effectiveness of genes and plot the pROC software package. The x-Cell software was used to calculate the enrichment score of 34 immune cells in each sample. Finally, four key genes (PSMA4, PSMA1, PSMB1, and PSMA3) were selected. Blood samples were analyzed using the qPCR method. Results:: A total of 373 immune-related differentially expressed genes were identified. Eight genes (PSMC6, PSMB2, PSMB1, PSMA3, PSMA4, PSMA1, PSMD7, and PSMB5) were screened from the 178 nodes and 596 edges as hub genes of the PPI network, which were significantly related to amino acid metabolism, hematopoietic stem cell differentiation, cell cycle, and immune processes. PSMA4, PSMA1, PSMB1, and PSMA3 were defined as immunological biomarkers for PBC with an AUC value of the ROC curve > 0.7. Immunoinfiltrating cell analysis showed that the proportion of eosinophils was significantly higher in PBC patients compared to the control group, whereas the proportion of CD4+ memory T cells, plasma cells, Th2 cells, and cDC cells was significantly lower in PBC patients than the control group. Plasma cells were associated with all four immunological biomarkers. Seven PBC patients and seven healthy subjects were selected for peripheral blood qPCR validation, which demonstrates that PSMB1, PSMA3, PSMA1, and PSMA4 levels were significantly lower in PBC patients than healthy subjects, with a statistically significant difference. Conclusion:: Bioinformatics screened eight key genes, of which four were key immunological markers and may serve as a basis for clinical diagnosis and mechanism exploration.

The role of GATA2 in adult hematopoiesis and cell fate determination.

The correct maintenance and differentiation of hematopoietic stem cells (HSC) in bone marrow is vital for the maintenance and operation of the human blood system. GATA2 plays a critical role in the maintenance of HSCs and the specification of HSCs into the different hematopoietic lineages, highlighted by the various defects observed in patients with heterozygous mutations in GATA2, resulting in cytopenias, bone marrow failure and increased chance of myeloid malignancy, termed GATA2 deficiency syndrome. Despite this, the mechanisms underlying GATA2 deficiency syndrome remain to be elucidated. The detailed description of how GATA2 regulates HSC maintenance and blood lineage determination is crucial to unravel the pathogenesis of GATA2 deficiency syndrome. In this review, we summarize current advances in elucidating the role of GATA2 in hematopoietic cell fate determination and discuss the challenges of modeling GATA2 deficiency syndrome.

A Study on Heterogeneity and Early Response to Chemotherapy in Pediatric ETV6-RUNX1 Positive Acute Lymphoblastic Leukemia By RNA-Seq Expression Profile

Objective ETV6-RUNX1 positive ALL is the most common type in childhood acute lymphoblastic leukemia(ALL), and it is also a type with good prognosis. However, there are still recurrent or refractory cases in this type, and there is still heterogeneity among individuals.In this research, we investigate the heterogeneity of ETV6-RUNX1 positive ALL in children by RNA-seq expression profile, and to explore the early response to chemotherapy and microenvironment characteristics of different clusters, so as to provide evidences for the exploration of genetic high-risk factors and clinical personalized diagnosis and treatment of such children. Method From September 2019 to June 2021, 72 children with newly diagnosed ETV6-RUNX1 positive ALL in standard risk group were enrolled and treated with CCLG-ALL-2018 chemotherapy protocol, which were all detected by RNA-seq technology. The sequencing results were analyzed by cluster analysis and bioinformatics analysis. We explored the early response to chemotherapy and microenvironment characteristics of the different clusters. Result ETV6-RUNX1 positive ALL were divided into three groups according to the RNA-seq expression profile including 16 cases (22.2%) in ER1 group, 35 cases (48.6%) in ER2 group and 21 cases (29.2%) in ER3 group. The number of patients with co-negative minimal residual disease (MRD) by flow cytometry (MRD-FCM) and next-generation sequencing (MRD-Gene) on the 15th day of chemotherapy in ER1 and ER3 groups was significantly different (3/16 vs. 13/21, P=0.0178), while there were 17 cases (17/35) with co-negative MRD in ER2 group after fifteen-day chemotherapy. It showed that the proportion of NK cells ( P=0.056) and CD8 + T cells ( P=0.047) in peripheral blood in ER1 group were both higher than those in ER3 group. Compared with ER3 group, ER1 group showed low expression of RPL41 and high expression of MRPS28 and SERPIND1, which were mainly involved in stress, proliferation and drug resistance of cells and the differentiation of hematopoietic stem cell. In addition, ER1 group showed lower expression of ribosome-related genes and higher activation of mTOR pathway than ER3 group. Conclusion The heterogeneity in gene expression profile were presented in children with ETV6-RUNX1 positive ALL. Compared with ER3 group, patients in ER1 group may have a poorer early response to chemotherapy with a relatively higher risk due to the stronger proliferation and drug resistance of leukemic cells probably. For patients in ER1 group, even if MRD is negative on the 15th day of themotherapy, it may be necessary to strengthen the observation of early response and consider the treatment for escalated risk in time.

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

Luspatercept Modulates Inflammation in the Bone Marrow, Restores Effective Erythropoiesis/Hematopoiesis, and Provides Sustained Clinical Benefit Versus Epoetin Alfa (EA): Biomarker Analysis from the Phase 3 COMMANDS Study

Introduction :Anemia is common in patients (pts) with myelodysplastic syndromes (MDS) due to ineffective erythropoiesis, characterized by increased dysplastic erythroid precursors (EPs) and abortive differentiation in the bone marrow (BM). Insufficient red blood cells (RBCs) lead to chronic anemia and heart failure due to oxygen deprivation. Luspatercept is approved to treat anemia after erythropoiesis-stimulating agent (ESA) failure in pts with transfusion-dependent, lower-risk MDS (LR-MDS). In the phase 3 COMMANDS trial (NCT03682536), 58.5% of ESA-naive luspatercept treated pts with LR-MDS experienced RBC transfusion independence for ≥ 12 wk with concurrent mean hemoglobin (Hb) increase ≥ 1.5 g/dL during wk 1-24, vs 31.2% of EA-treated pts. Aims :To differentiate the mechanism of action of luspatercept from EA and determine its correlation with clinical benefit in the COMMANDS study. Methods :Cytomorphology assessments were performed on BM aspirates at baseline, wk 24 and 48. Complete blood count, Hb, and reticulocytes (retics) were measured in peripheral blood. BM mononuclear cells (BMMC) were subjected to bulk RNA-seq. Serum cytokine analysis was performed at screening and wk 24 using Rules-Based Medicine's Human Inflammation MAP ® v.1.1 and a custom Human MAP ELISA panel. Results :In ITT population, EPs, retics, and Hb were increased vs baseline in the luspatercept arm at wk 24 ( P < 0.001) and wk 48 ( P < 0.005). EPs, retics, and Hb were increased in the EA arm at wk 24 ( P < 0.01), but this was not sustained to wk 48. Subgroup analysis showed similar trends for ring sideroblast (RS)+ pts in both arms. While RS− pts in the EA arm showed similar trends to the ITT population, luspatercept RS− pts showed trends of EP expansion post-wk 24 only. Retics increased significantly in RS− luspatercept pts at wk 24 ( P < 0.005). Hb increased more significantly at wk 24 with EA ( P < 0.003) but at wk 48 with luspatercept ( P < 0.005).Gene set enrichment analysis of BMMC revealed enrichment of early, mid, and late EP genes at baseline favored response to luspatercept, but enrichment of mid and late EP genes was unfavorable for EA (Figure A). Significant downregulation of REACTOME IL-1 signaling (normalized enrichment score [NES] = −2.27; adjusted [Adj] P < 0.0001 [vs EA = not significant (ns)]), BIOCARTA IL-6 pathway (NES = −1.99; Adj P = 0.0025 [vs EA = ns]) and IFNα response (NES: −2.26; Adj P < 0.0001 [vs EA = ns]) was observed with luspatercept. BIOCARTA IL-1R pathway was more significantly reduced with luspatercept (NES = −1.78; Adj P = 0.0073) compared with EA (NES = −1.79; Adj P = 0.0172), as was IL-10 signaling (luspatercept NES = −2.39; Adj P < 0.001 vs EA NES = −1.82; Adj P = 0.0125). TGF-β, apoptosis, and spliceosome pathways down regulation was also observed with luspatercept. At wk 24, luspatercept responders had a median 640 pg/mL decrease ( P = 0.0035) of N-terminal pro-brain natriuretic peptide (NT-proBNP), produced by the heart, compared with a median 360 pg/mL decrease in EA arm responders ( P = 0.37). At wk 24, non-responders had a median increase of 775 pg/mL ( P = 0.0032) in the EA arm and 31.5 pg/mL ( P = 0.95) in the luspatercept arm. Conclusions : Multiplatform analysis of primary BM samples from a phase 3 trial of luspatercept revealed novel response-associated biomarkers. Compared with EA, luspatercept led to sustained increase of EPs and retics over 48 wk with a concomitant Hb increase. RS− pts showed Hb accumulation peaked at wk 24 in the EA arm. Conversely, luspatercept in RS− pts significantly increased retics at wk 24 and EPs past wk 24 with concomitant peaking of Hb at wk 48, suggesting gradual and sustained clinical benefit. Unlike EA, which plays a role in early differentiation of hematopoietic stem cells (HSCs), luspatercept acts on different erythroid stages, leading to EP expansion and maturation. Furthermore, downregulation of IL-1, IL-1R, IFNα, IL-6 pro-inflammatory (Figure B), and IL-10 anti-inflammatory signaling with luspatercept suggests modulation of the BM inflammatory environment in MDS to restore HSC function for normal hematopoiesis (Wang J, et al. Front Immunol 2022;13:951937). Additionally, decreased or static levels of NT-proBNP with luspatercept suggest a positive effect in mitigating cardiac damage. These novel insights obtained from analysis of a large controlled clinical trial mechanistically differentiate the superior clinical benefit of luspatercept from EA in treating anemia due to MDS.

From in silico to in Vitro to In Vivo: Increasing Megakaryocytes to Treat Thrombocytopenia

Hematopoietic stem cells undergo a multi-step differentiation program through several precursor stages to give rise to megakaryocytes (Mk), which ultimately release platelets. Thrombocytopenia can manifest in various clinical settings and can adversely affect a patient's quality of life through easy bruising and risk of bleeding. An example is chemotherapy-induced thrombocytopenia (CIT) which occurs when myelosuppressive chemotherapy is given, especially with certain agents such as platinum analogs and cyclophosphamide. We used a single-cell RNA-sequencing approach to study the differentiation of hematopoietic stem cells to Mk progenitors (MkP) and delineated the trajectory that defines the transition of cells to the megakaryocytic lineage. Using our proprietary platform which pairs new approaches in computation and machine learning in drug discovery, we predicted small molecule interventions that could modulate the hematopoietic lineage direction to increase the differentiation towards MkP and subsequent increases in platelet counts. Using an in vitro 5-day CD34-based Mk differentiation assay we demonstrated that some of our predicted interventions increased generation of MkP from stem cells. Analysis of early MkP (CD34-CD235a-CD71+CD41b+CD42b-) and late MkP (CD34-CD235a-CD71-CD41b+CD42b+) by flow cytometry elucidated differential mechanisms of action such as induction of early or late MKP or both. We performed an 8-day study in healthy mice, which revealed that a selected set of compounds increased MkPs in vivo, consistent with our predicted cell behavior and in vitro findings. To evaluate a possible application for this MkP and platelet increases we tested the efficacy of one of our hit compounds CP3 in a CIT mouse model using Carboplatin. In this model, we recorded a >70 % reduction in platelet counts on day 9 post carboplatin administration, with platelet numbers recovering on day 13 and return to basal levels by day 21. To assess the efficacy of CP3, mice dosed first with this compound were challenged with carboplatin. The primary endpoint of this study was the ability of the compound to rescue mice from nadir of thrombocytopenia and accelerate recovery to baseline. Prior to carboplatin dosing, 7 days of CP3 dosing showed a dose dependent increase in platelet counts confirming our initial studies. However, CP3 dosing did not rescue platelet nadir or accelerate recovery to baseline in the CIT model. This study demonstrated the capacity of CP3 to increase platelets in a healthy BM environment but not under conditions of myelosuppression as seen in the CIT mouse model. Taken together, these findings highlight the benefits of our platform to predict compounds that modulate lineage differentiation towards MkP, and a resultant increase in platelet numbers in an animal model.

A Phase 2 Study of Canakinumab in Patients with Lower-Risk Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background Myelodysplastic syndromes (MDS) lie on a continuum of myeloid neoplastic progression between clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Interleukin 1 beta (IL-1β)-induced inflammasome activation is a mechanism of cancer initiation and progression. Given that IL-1β upregulation induces aberrant myeloid differentiation of hematopoietic stem and progenitor cells (HSPCs), we hypothesize that IL-1β plays a critical role in MDS pathogenesis, and targeting IL-1β signaling will overcome aberrant MDS HSPCs' myeloid skewing. Here, we report final results of a phase 2 study evaluating the safety and efficacy of canakinumab, an IL-1β inhibitor, in patients with lower-risk MDS or chronic myelomonocytic leukemia (CMML). Methods We conducted a single center, open-label phase 2 trial at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS/CMML who have previously been treated were included in the study. Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. The primary objective was to determine the clinical activity of canakinumab in MDS/CMML patients. Response was assessed using the modified International Working Group 2006 criteria for MDS (Cheson et al. 2006). Secondary objectives were to evaluate the safety profile including rate of transfusion independence (TI), duration of response (DoR), progression-free survival (PFS), and overall survival (OS). All patients who received 1 dose of study drug were included in the analyses. This study is registered with ClinicalTrials.gov (NCT04239157). Findings Between August 2020 and May 2023, 27 patients were enrolled in the phase 2 portion of this study, and 2 patients failed screening. The median age was 74 years with 15 (60%) male patients; 20 patients (80%) were post-hypomethylating agent failure with a median number of prior lines of therapy of 2 (1-5). Transfusion dependency was observed in 24 patients (96%) prior to canakinumab initiation. Fourteen (56%) patients showed normal karyotype. IPSS-R stratification revealed an intermediate-2 risk in 12 (48%) patients and a high risk in one (4%). The most common mutations were SF3B1 (40%), TET2 (32%), DNMT3A (28%), and RUNX1 (24%). IPSS -M risk score was calculated for 24 cases showing one (4%) very low (VL), four (16%) low (L), 14 (16%) moderate low (ML), eight (32%) moderate high (MH), five (20%) high (H), and two (8%) very high (VH) risk categories. Canakinumab was well tolerated and no drug-related toxicities were observed. One death due to sepsis, which was deemed not treatment-related, occurred on the study drug. Out of 23 evaluable patients, the overall response rate was 17·4%, with erythroid and platelet hematological improvement (HI-E and HI-P, respectively) confirmed in 3 (13.0%) patients and 1 (4.3%) patient, respectively. Thirteen patients had stable disease (56·5%) and 6 (26·1%) progressed during therapy, 1 of which transformed to AML (Fig. 1). TI was achieved in 3 patients (median DoR 8·53 months (95% CI 0·41-16·1) and 2 of them maintained TI for over 12 months. With a median follow-up time of 22·6 months (95% CI 15·0-29·4), median OS was 17·3 months (95% CI 14·3-not estimable). We performed separate univariate analyses to evaluate any associations between the IPSS-M and OS/PFS. The median OS in patients with higher-risk MDS by IPSS-M (MH, H, VH) was 15·0 months vs 29·4 months in the lower-risk disease by IPSS-M (VL, L, ML) group (p=0·12). Interestingly, statistically significant findings were observed with 1-year PFS when stratifying patients into higher vs lower risk MDS by IPSS-M (64·3% vs 100·0%, respectively; p=0·022, Fig. 2). Conclusion In this cohort of MDS patients who had experienced multiple lines of prior therapy and exhibited high-molecular-complexity, canakinumab showed limited efficacy (HI 17.3%). Nevertheless, canakinumab showed a good safety profile and yielded sustained long-term responses in patients with single somatic driver mutation in TET2 or DNMT3A. This suggests that clonal complexity, and therefore disease burden, may be a determining factor in response to canakinumab. Therefore, we have amended the protocol of a phase 1/2 clinical trial evaluating the safety and activity of canakinumab in clonal cytopenia of unknown significance (CCUS) and low-molecular complexity MDS patients. Our results will clarify the role of IL-1β signaling in MDS initiation and progression.

BGS-2456 Is a Novel Potent Covalent Inhibitor of FLT3 That Highly Discriminates Against KIT and Is Not Toxic Toward Normal Hematopoiesis in Vitro

Introduction: Class III receptor tyrosine kinases include FLT3, KIT, PDGFRA/B and CSF1R. Activating mutations in FLT3 represent the most common genetic anomalies in AML (30%). While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition. Preclinical studies suggest that coinhibition of FLT3 and KIT can result in non-specific myelosuppression, which can limit dose intensification and the ability to safely achieve maintenance dosing. A potent FLT3 TKI that is non-myelosuppressive may enable prolonged treatment in conjunction with chemotherapy and thereby substantially improve clinical outcomes, similar to BCR::ABL1 TKIs in Ph+ ALL (Slayton et al, JCO 2018). FF-10101 is a recently described covalent inhibitor to irreversibly bind FLT3. In vitro and in vivo studies have revealed that FF-10101 exhibits substantial activity against FLT3 and resistant mutants F691L and D835V/Y. However, it does not effectively discriminate against KIT inhibition (Yamara et al, Blood 2018). In clinical experience, a majority of responses were associated with persistent myelosuppression (Levis et al, JCO 2021 39:15_suppl, 7008). Further clinical development of FF-10101 has been abandoned. Here we describe a novel covalent inhibitor (BGS-2456) that potently targets FLT3 with outstanding selectivity against KIT. Results: To confirm that BGS-2456 is a covalent FLT3 inhibitor, we tested various Cys→Ser substitutions in FLT3-ITD. Of these, only substitution of C695 resulted in loss of activity, suggesting that this residue is critical for covalent bond formation with BGS-2456, as has also been demonstrated for FF-10101 (Yamara et al, Blood 2018). Additionally, the compound without the covalent warhead (BGS-2457) was found to be unimpacted by the C695S substitution and substantially less potent than BGS-2456 (Figure). To characterize in vitro properties of BGS-2456 and whether it achieves selectively against FLT3 relative to KIT, we performed cell viability assays of the patient-derived FLT-ITD-positive AML Molm14 cell line as well as the gastrointestinal stromal tumor cell line GIST-T1, which contains an activating juxtamembrane mutation in KIT. We compared EC50 ratios with other FLT3 TKIs and found that BGS-2456 is highly potent, with an EC50 value of ~0.2nM. Moreover, BGS-2456 displayed remarkable selectivity toward FLT3, >10-fold greater than all other TKIs tested. Kinome profiling confirmed FLT3 as the kinase that was most highly inhibited by BGS-2456 among the 300 kinases assessed, with BMX being a distant second (Figure). To assess whether the selectivity of BGS-2456 results in less myelosuppression in vitro, we performed CFU assays of healthy bone marrow. Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells. Finally, we assessed BGS-2456's ability to retain activity against the known clinically problematic drug-resistant FLT3-ITD mutants D835Y and F691L. Encouragingly, BGS-2456 retained activity against both D835Y and F691L mutants in the low nanomolar range. Conclusion: This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

High Frequency of RUNX1 Mutation in Acute Myeloid Leukemia at Puerto Rico

Acute Myeloid Leukemia (AML) is the most common form of acute leukemia in adults, with a median age at diagnosis of 68 years. Recurrent genomic abnormalities are established as diagnostic, prognostic markers, and play an essential role in leukemogenesis. For instance, loss-of-function mutations of the runt-related transcription factor-1 (RUNX1) have been associated with distinct clinical and genetic features and overall adverse risk in adult AML. Lack of a specific targeted therapy and resistance to standard intensive chemotherapy likely accounts for the poorer outcome observed in AML expressing RUNX1. Additionally, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies. Previuos studies has identified mutant RUNX1 in 5-10% of patients at a median age of 68 years old. The objective of our retrospective study was to identify the incidence and characteristics of RUNX1 mutations in our population. RUNX1 plays an important role in the regulation and differentiation of normal hematopoietic stem cells. Therefore, intragenic mutation of RUNX1 leads to impaired hematopoiesis and ultimately leukemia. The study examined newly diagnosed (ND) patients-AML with RUNX1 mutation by NGS; that were at least 18 years of age or older with cytogenetic and molecular analysis in a period of 4 years (2019-2022). According to the Puerto Rico Cancer Registry from 2015 to 2019 a total of 551 cases of AML were reported. All data regarding demographic, bone marrow biopsy, cytogenetics, and molecular markers were obtained from electronic medical record at San Juan City Hospital with institutional review board approval in acordance with the Declaration of Helsinki. The San Juan City Hospital is the major referral center on the island for initial evaluation and management of AML. A total of 157 patients with ND AML were identified during the study period. 58% female and 42% male with a median age of 57 years old at time of diagnosis. Compared to an expected ~5-10% incidence of RUNX1 mutations, our study population revealed the presence of RUNX1 in 24% of our AML patients, with a median age less than 60 years old. More specificially within the 37 patients with RUNX1 mutations, 46% female and 54% male with a median age of 56 years old with an average variable allele frequency(VAF) of 40%. Complex cytogenetics was identified in 41% of RUNX1 cases. Co-occuring mutation of RUNX1 with TP53 was identified in 19% of patients. Our study showed a high expression of RUNX1 in Puerto Rican ND-AML. The median age at diagnosis and the average VAF suggest the possibility of a germline RUNX1 in our population. Previous studies in medical literature have identified a correlation for germline RUNX1 in patients diagnosed before 60 years old and with a VAF above 40%. Future studies with larger sample size and longer follow-up may confirm if there is a germline predisposition in our patients for RUNX1.

Establishment of Human Bone Marrow-on-Chip As a Preclinical Model to Evaluate Drug-Induced Toxicities and Myelofibrosis Patient-Specific Pathophysiology

Hematopoiesis takes place in the bone marrow (BM) and is supported by a complex cellular and molecular network in the BM microenvironment. The inaccessibility of living human BM hampers the study of its pathophysiology under stress induced by drugs, radiation, or genetic mutations. Commonly used models of the human BM microenvironment rely on mouse models and 2D or 3D static tissue cultures. Although existing in vitro hematopoiesis models have led to advances in the field, they fail to recapitulate the biology of cellular interactions, cell extrinsic factors, and complex functions of living human BM hematopoiesis. This has also limited our understanding of human BM biology and the influence of BM niche cells in BM defects in diseases such as myeloproliferative neoplasms (MPNs) and leukemias. Consequently, there is an unmet need to develop a preclinical human BM hematopoiesis model that supports growth and differentiation of multilineage hematopoietic stem cells to enable evaluation of investigational compounds for myeloerythroid toxicity and efficacy. Microphysiologic systems (MPS), such as organ-on-chips, emulate the complex interplay between intrinsic and extrinsic cell factors in the BM microenvironment, the 3D tissue architecture, and are more clinically relevant compared with static co-culture approaches (PMID: 31988457; PMID: 37359774). Here we present the development of a human BM-on-chip MPS model to recapitulate both healthy and MPN disease and evaluate the alterations in the BM niche environment. We utilized a commercially available organ-on-chip microfluidic platform (Emulate, Boston, MA) to develop a living human BM with a functional hematopoietic niche in vitro by perfusion with culture medium. This fluidic-controlled modular chip comprises an osteogenic apical channel separated from a vascular basal channel by a semiporous membrane, which allows for media perfusion, cellular crosstalk, and intercellular signaling. The osteogenic apical channel is filled with a fibrin gel that allows the co-culture of all critical cells of the BM niche, including osteoblasts, endothelial cells, mesenchymal stem cells, hematopoietic stem and progenitor cells. The parallel basal channel is lined by human vascular endothelium and is perfused with culture medium optimized to allow survival and development of functional hematopoietic lineage niche cell populations. This unidirectional fluidic MPS model allows for collection of cell effluents for evaluation of cell-extrinsic factors such as cytokines, extracellular vesicles, and metabolites. The modular and accessible nature of this system provides an advantage to investigate the effects of the microenvironment and drug candidates individually on the osteogenic and vascular compartments. This human BM-on-chip has been tested with cells from several healthy and myelofibrosis donors. Utilizing imaging, flow cytometry, cytokine profiling, and transcriptomics, we demonstrate that the vascularized human BM-on-chip recapitulates the living human hematopoietic niche, with BM relevant cytokine levels, retains hematopoietic stem and progenitor cells in in vivo-like proportions for over 3 weeks in culture, allows for the differentiation of BM niche lineage cell populations similar to human BM aspirate, and captures human donor variabilities. We also show that BM-on-chips derived from MPN patients recapitulate the hematopoietic defects to understand the biology and disease response in myelofibrosis. Our robust experimental workflow and bioinformatics platform will be utilized to evaluate candidate compounds for BM myelotoxicities, drug mechanisms of action, and biomarkers in a multifactorial manner to support preclinical programs. This biomimetic platform offers a new approach for analysis of drug responses and toxicities in BM as well as for study of hematopoiesis and hematologic diseases such as myelofibrosis.

Potentiality of Bone Marrow-Derived Mesenchymal Stromal Cells (BM-MSCs) to Differentiate into the Osteogenic Lineage in in Vitro Model of Tissue Regeneration

Introduction:The mesenchymal stromal cells (MSCs) have gained considerable popularity owing to the vast possibilities and lack of ethical constraints. MSCs serve as multipotent progenitors for several niche components in the bone marrow (BM) and play an essential role in the quiescence, proliferation, and differentiation of hematopoietic stem cells. Recently, MSCs from BM (BM-MSCs) represent an attractive cell source for tissue engineering, thanks to their immunomodulatory property, self-renewing and high proliferative capability. BM-MSCs have multi-lineages (adipogenesis, chondrogenesis, osteogenesis) potential and in combination with biomaterials, well support cell-attachments and stimulate extracellular matrix synthesis. For these reasons, BM-MSCs have a great potential for regenerative medicine applications. The present research aims to evaluate the ability of BM-MSCs to proliferate and differentiate toward the osteogenic lineage into innovative three-dimensional bioresorbable scaffold based on gelatin-chitosan hydrogel with a poly(lactic acid) lattice structure (PLA-CH) for regenerative medicine applications in the presence of fetal bovine serum (FBS) or human platelet lysate (HPL) with or without the osteogenic medium (OM). Methods: BM-MSCs were expanded to the passages 3 or 4 and seeded into the PLA-CH, in dry state, at a cellular density of 7x10 5 cells/scaffolds in growth medium (GM). For inducing osteogenic differentiation, cells/scaffolds constructs were cultured in 24-well plates for 4 weeks with OM consisting of a high-glucose DMEM supplemented with 10% FBS or 5% HPL, 10 −7M Dex, 25 mg/ml l-ascorbic acid, and 3mM NaH 2PO 4. For histomorphological analysis at optical microscope, scaffolds were embedded in paraffin using an automatic processor Donatello series 2 (Diapath S.p.A., Bergamo, Italy). Serial paraffin sections (5 µm thick) of each sample were cut, deparaffinized, and rehydrated, according to standard procedures and stained with hematoxylin-eosin stain for general morphology and Von Kossa (Bio-Optica, Milan, Italy) for calcium deposition. Immunohistochemistry for the osteogenic marker Osteocalcin (OSC) (Santa Cruz Biotechnologies, USA) was also performed. For Scanning Electron Microscopy (SEM) (EVO LS-10 manufactured by ZEISS) observation, samples have been progressively dehydrated through immersion in alcohol solutions. The local changes in elemental composition were investigated using the Energy Dispersive X-ray (EDX) analyzer. Results: The results showed that BM-MSCs have higher affinity to attachment, migrate and differentiate toward the osteogenic lineage inside the novel scaffold PLA-CH both with HPL and FBS as supplement in the culture media. The use of HPL for cell expansion offers the possibility to obtain a safer cellular products without xenogenic contaminants. Cells develop a large spreading area with elongated fibroblast-like morphology preferentially inside the hydrogel pores, showing great affinity with the scaffold. This is confirmed also by histomorphological analyses at optical microscope, showing homogeneous colonization of cells inside the hydrogel pores (figure 1a). The results showed the presence of calcium deposits in the mash of the scaffold with Von Kossa stain (figure 1b), moreover cells appeared well integrated with a positivity for OSC showing a clear differentiation state as osteoblasts (figure 1c). Calcium and phosphorous were detected with SEM-EDX on the samples incubated in osteogenic medium (figure 1 d,e). Conclusion: MSCs, derived from bone marrow, associated with scaffold PLA-CH proved to be biocompatible and promising for personalized medicine. In fact, BM-MSCs were able to growth, colonize and osteo-differentiate throughout the hydrogel, demonstrating their potential application for bone regenerative medicine. Figure 1. Histomorphological analysis at the optical microscope of PLA-CH with BM-hMSCs in the GM at 100 m m ( a), Von Kossa stain showing calcium deposit distribution on PLA-CH with BM-hMSCs in the OM at 30 m m ( b), immunohistochemistry for OCN with BM-hMSCs differentiated to osteoblast in the OM at 10 m m ( c). Evaluation of calcium (Ca) and phosphorous (P) with SEM-EDX of PLA-CH with BM-hMSCs in OM day 28 ( d,e).

Sting-Mediated Interferon Signaling Exerts Potent Antileukemic Effects in High-Risk Myeloid and Monocytic Malignancies

Clinical outcomes of patients (pts) with high-risk (HR) myeloid neoplasms remain dismal despite available therapies. Interferons (IFN) regulate hematopoietic stem-progenitor cell (HSPC) differentiation, proliferation and self-renewal capabilities and preferentially impair clonal HSPC fitness. To evaluate if myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) HSPCs activate IFN responses we performed RNA sequencing in bone marrow (BM) CD34 + cells obtained from pts with MDS (n=58), CMML (n=27) and age-matched healthy donors (HDs, n=17). This analysis revealed significant deregulation of IFN pathway genes in HSPCs from pts with CMML and to a lesser extent in those of pts with MDS. Activation of the stimulator of interferon genes (STING) can inhibit stem-cell function and induce myeloid and monocytic cell death via IRF3-dependent and independent mechanisms. Therefore, we hypothesized that STING pathway activation might have therapeutic potential in high risk (HR) myeloid and monocytic (MyMo) neoplasms. In vitro treatment of monocytic MOLM13 and THP1 cell lines with the novel synthetic STING agonist dazostingag resulted in induction of IRF3 phosphorylation, caspase 3 cleavage and apoptosis. Dazostingag was also able to induce enhanced cell death in multiple myeloid cell line cultures. Given the ability of dazostingag to induce apoptosis of monocytic cell lines, we sought to evaluate the activity of monocyte-directed STING activation using TAK-500, an IgG1 anti-CCR2 antibody linked to dazostingag as a STING agonist payload. To do so, we first analyzed the expression of the monocyte-specific cysteine-cysteine chemokine receptor 2 (CCR2) in primary pts samples and cell lines. Both CCR2 RNA and protein expression levels were higher in CMML compared to HDs or MDS. In accordance with this finding, monocytic cell lines (MOLM13, THP1) had significantly higher CCR2 expression compared to non-monocytic cell lines (SKM1, HL60, U937, KG1). Treatment of MOLM13 and THP1 cell lines with TAK-500 induced strong IRF3 phosphorylation, caspase 3 cleavage and apoptosis. In vitro treatment of BM mononuclear cells (MNCs) from pts with MDS (n=10), CMML (n=11) or AML (n=8) in co-culture with mesenchymal stromal cells with TAK-500 (0.5 mM) or dazostingag (1 mM) resulted in increased cell death of MNCs from pts with CMML and AML. TAK-500 (0.5 mM) significantly depleted CCR2 + BM MNCs from all disease groups but did not affect CCR2 + cells from HDs. Next, to evaluate whether STING activation impairs repopulation potential of neoplastic HSPCs we performed colony-formation studies in CD34 + BM cells from pts with CMML (n=8), MDS (n=3) and AML (n=4). Treatment with TAK-500 (0.5mM) and dazostingag (1mM) resulted in significantly impaired myeloid colony-formation in all disease groups without significantly impairing erythroid colony-formation in MDS or CMML samples. Taken together, these data suggests that STING activation might induce aberrant myelomonocytic cell death and deplete aberrant HSPCs in HR-MyMo neoplasms. To evaluate the in vivo efficacy of TAK-500 and dazostingag, we established xenograft models by inoculating NOG-IL15 transgenic mice with MOLM13 cells. Treatment with TAK-500 and dazostingag resulted in reduction of tumor burden evaluated by bioluminescence with no observed toxicities. Administration of weekly doses of TAK-500 induced enhanced and sustained tumor burden suppression. To determine the in vivo effect of STING activation in an immune competent model of CMML, we then evaluated the effects of TAK-500 and dazostingag in Vav-KDM6B/Tet2 f/f/Vav-Cre mice, a previously reported (Wei et al Leukemia 2022) animal model with CMML phenotype. Treatment with TAK-500 led to enhanced apoptosis of Ccr2 + BM cells in these mice. Moreover, the number of colonies formed by lineage negative/Sca1 positive/cKit positive BM progenitor (LSK) cells isolated from TAK-500 treated mice was significantly lower than in vehicle treated control mice, suggesting a negative regulation of repopulating capacity of Tet2 deficient/ KDM6B overexpressing BM HSPCs by TAK-500. Evaluation of the efficacy of TAK-500 and dazostingag in patient-derived xenograft models of primary CMML samples is planned. Taken together, our data suggests that CCR2-directed STING activation might have therapeutic potential in HR-MyMo neoplasms by depleting CCR2 + cells and impairing clonal HSPC repopulation potential.

TIF1γ Counteracts Ferroptosis to Drive Erythroid Progenitor Differentiation

Understanding in-vivo mechanisms of hematopoiesis is critical for developing directed blood differentiation approaches to treat blood disorders such as leukemias. Zebrafish moonshine ( mon) mutant embryos defective for transcriptional intermediary factor 1 gamma ( tif1γ), a conserved transcription elongation and chromatin factor, lack red blood cells due to a block in hematopoietic stem cell differentiation along the erythroid lineage. We recently showed that TIF1γ plays a critical role in mitochondrial metabolism, including maintaining adequate coenzyme Q levels. Through a chemical suppressor screen for the mon mutant, we identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase to promote erythroid differentiation in mon mutants due to its functional link to the electron transport chain. In agreement, our in-vivo metabolomics analyses identified nucleotide metabolism as the most significantly altered processes in mon mutants, with elevated levels of uridine monophosphate and low levels of N-carbamoyl-L-aspartate. Interestingly, imbalances in nucleotide metabolism similar to those in mon mutants have also been reported in the presence of active ferroptosis, a pathway of programmed cell death due to iron-dependent lipid peroxidation. Through transcriptome profiling upon tif1γ loss of function in zebrafish embryos at the onset of hematopoiesis, we uncovered an expression signature indicative of activated ferroptosis. gpx4 and fsp1, inhibitors of ferroptosis acting in parallel pathways, were both downregulated, and chac1 and ptgs2, an enhancer and marker of ferroptosis, respectively, were upregulated. In addition, parallel genome-wide expression and chromatin immunoprecipitation analyses identified anti-ferroptotic genes as direct TIF1γ targets in human cells. In support of these results, we found mon mutants to exhibit increased levels of lipid peroxidation. Functionally, tif1γ loss of function synergizes with loss of gpx4 in blocking erythroid differentiation. These results demonstrate a tight coordination of nucleotide metabolism, mitochondrial respiration, and the regulation of lipid peroxidation as a key function of tif1γ-dependent transcription that drives cell fate decisions in the early erythroid lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases.

Quantity and Functional Exhaustion of Mucosal-Associated Invariant T Cells in Acute Myeloid Leukemia: Friends or Foes?

Abstract Introduction Acute Myeloid Leukemia (AML) is a hematological malignancy affecting hematopoietic progenitor cell differentiation, proliferation, and cell cycle progression, possibly involving disturbances in host immune function. Comprehensive understanding of the immune system composition and function in AML patients is crucial for developing novel immunotherapeutic strategies. Mucosa-Associated Invariant T cells (MAITs), as a subset of unconventional T cells, bridge innate and adaptive immunity and have been predominantly studied in the context of mucosa-related cancers. However, their role in AML remains largely unexplored. Investigating the association between MAITs and AML is of significant importance for understanding the immune escape mechanisms in AML, developing novel immunotherapeutic approaches, and predicting patient prognosis. Methods This study aimed to delineate the longitudinal immune profile of MAITs during AML pathogenesis and treatment, which may contribute to understanding immunological changes of MAITs in AML and predicting patient prognosis. In this prospective study, we collected peripheral blood or bone marrow samples from 131 AML patients, including 99 newly diagnosed cases, 18 in remission, and 14 in relapse, as well as 69 healthy controls. We performed multi-parameter flow cytometry for phenotypic and functional analysis of MAITs and explored their relationship with disease features, progression, and prognosis. Results We initially demonstrated that MAITs reflected immunological defects in AML patients. MAITs showed signs of depletion in both frequency and absolute count at initial diagnosis (p<0.0001) (Fig. 1A), especially in the CD8 + MAITs subset. The frequency of MAITs reflected AML cell genetics, tumor burden, disease status, and treatment responsiveness (Fig. 1B). AML patients exhibited reduced frequency of effector memory MAITs (p<0.0001) and increased frequency of terminal differentiated MAITs (p<0.0001) in peripheral blood (Fig. 1C), indicating altered reactivity of MAIT cells to antigen stimulation. MAITs displayed a state of high activation and even exhaustion, as evidenced by upregulated PD-1 expression. Impaired secretion of Th1-type cytokines and increased secretion of Th17-type cytokines (Fig. 1D), granzyme B, and perforin were observed in MAITs, while MAITs also shifted towards secreting tumor-promoting cytokines such as IL-8. Lower frequency of MAITs was correlated with inferior Overall Survival (OS) and Progression-Free Survival (PFS) (Fig. 1E). Multivariate analysis identified MAITs frequency <2.12% as an independent prognostic factor affecting OS. Conclusion Our study highlights the role of MAITs in immunological defects in Acute Myeloid Leukemia and reveals their functional impairment and enhanced tumor-promoting effects. The frequency of MAITs in peripheral blood reflects the genetic features, tumor burden, disease status, and treatment responsiveness of AML patients. Lower frequency of MAITs is associated with inferior OS and PFS, serving as a prognostic indicator for AML.

Bone Marrow as a Therapeutic Target for Type 2 Diabetes Complications.

Type 2 diabetes mellitus (T2DM) is a world epidemic with a high prevalence and mortality. The origin of macro and microvascular complications associated with T2DM is complex and new mechanisms to explain their development are emerging. The changes induced by T2DM in the microenvironment of bone marrow (BM) alter the expansion and differentiation of stem cells and have been related to the development of micro and macrovascular diseases. Alterations in the differentiation and function of hematopoietic, endothelial, and mesenchymal stem cells in T2DM patients reduced the mobility of BM stem cells to the circulation and some immature, dysfunctional, or inflammatory cells pass to the blood (mobilopathy). Consequently, tissue repair is impaired, and the tissue damage caused by hyperglycemia, oxidative stress, and inflammation is increased. These alterations can contribute to diabetic complications, decreasing the quality of life, and increasing mortality. The modulation of the bone marrow microenvironment may be a therapeutic target for treating T2DM and its complications. This article analyses the changes induced in BM and their impact on the development of cardiovascular and kidney complications in T2DM. Also, different therapeutic strategies to restore the bone marrow microenvironment and function through the modulation of oxidative stress, inflammation, and adipogenicity are discussed, considering bone marrow as a novel potential therapeutic target to treat vascular complications of diabetes.

MAPK/ERK and PI3K/AKT signaling pathways are activated in adolescent and adult acute lymphoblastic leukemia.

The mitogen-activated protein kinase (MAPK)/ERK signaling cascade and the phosphoinosytol-3 phosphate/Akt (PI3K/Akt) pathways are involved in proliferation and differentiation of hematopoietic cells. The frequency of PI3K/Akt and MAPK pathway activation in adult acute lymphoblastic leukemia (ALL) still need to be elucidated. To assess the activity and prognostic implications of MAPK/ERK and PI3K/Akt pathways in adult (ALL). We examined 28 precursor-B-cell ALL and 6 T-cell primary ALL samples. Flow cytometry was employed to analyze the expression levels of phosphorylated ERK and phosphorylated Akt. Ten out of 15 (67%) ALL fresh samples (7 B-cell, 3 T-cell) showed constitutive p-ERK expression. The p-ERK mean fluorescent index ratio (MFI (R)) showed a tendency to be higher in ALL than in normal T lymphocytes (1.26 [0.74-3.10] vs. 1.08 [1.02-1.21], respectively [p = .069]) and was significantly lower than in leukemic cell lines (median MFI (R) 3.83 [3.71-5.97] [p < .001]). Expression of p-Akt was found in 35% (12/34) (10 B-cell, 2 T-cell). The median MFI (R) expression for p-Akt in primary blast cell was 1.13 (0.48-9.90) compared to 1.01 (1.00-1.20) in normal T lymphocytes (p = ns) and lower than in leukemic cell lines (median MFI (R) 2.10 [1.77-3.40] [p = .037]). Moreover, expression of p-ERK was negatively associated with the expression of CD34 (1.22 [0.74-1.33] vs. 1.52 [1.15-3.10] for CD34(+) and CD34(-) group, respectively, p = .009). Our findings suggest that both MAPK/ERK and PI3K/Akt are constitutively activated in adult ALL, indicating a targeted therapy potential for ALL by using inhibitors of these pathways.