Hematology
Abstract
SLFN14-related thrombocytopenia is a rare bleeding disorder caused by SLFN14 mutations altering hemostasis in patients with platelet dysfunction. Schlafen (SLFN) proteins are highly conserved in mammals where SLFN14 is specifically expressed in megakaryocyte (MK) and erythroblast lineages. The role of SLFN14 in megakaryopoiesis and platelet function has not been elucidated. We generated a new murine model with a platelet- and MK-specific SLFN14 deletion using platelet factor-4 (PF4) cre-mediated deletion of exons 2 and 3 in Slfn14 (Slfn14;PF4-Cre) to decipher the molecular mechanisms driving the bleeding phenotype. SLFN14;PF4-Cre+ platelets displayed reduced platelet signaling to thrombin, reduced thrombin formation, increased bleeding tendency, and delayed thrombus formation as assessed by intravital imaging. Moreover, fewer in situ bone marrow MKs compared to controls. RNA sequencing and gene ontology analysis of MKs and platelets from Slfn14;PF4-Cre homozygous mice revealed altered pathways of ubiquitination, ATP activity, cytoskeleton and molecular function. In summary, we investigated how SLFN14 deletion in MKs and platelets leads to platelet dysfunction and alters their transcriptome, explaining the platelet dysfunction and bleeding in humans and mice with SLFN14 mutations.
Authors
Rachel J. Stapley, Xenia Sawkulycz, Gabriel H.M. Da Mota Araujo, Maximilian Englert, Lourdes Garcia-Quintanilla, Sophie R.M. Smith, Amna Ahmed, Elizabeth J. Haining, Nayandeep Kaur, Andrea Bacon, Andrey V. Pisarev, Natalie S. Poulter, Dean P.J. Kavanagh, Steven G. Thomas, Samantha J. Montague, Julie Rayes, Zoltan Nagy, Neil V. Morgan
Jab1 promotes immune evasion and progression in acute myeloid leukemia models under oxidative stress
Abstract
Acute myeloid leukemia (AML) is the most common hematological malignancy. Leukemia stem cells exhibit high levels of oxidative stress, with reactive oxygen species (ROS) being the primary products of this stress, inducing the expression of Jab1. Previous studies have demonstrated that Jab1, as a transcriptional coactivator of c-JUN, promotes the malignant progression of AML under oxidative stress. However, its role in immune evasion is still under investigation. Here, we observed that knocking out Jab1 reduced the expression of immune checkpoints in vivo, effectively overcame the immune evasion of AML. Interestingly, the deletion of Jab1 had no impact on the maturation of normal hematopoietic cells in mice. Mechanistically, Jab1 directly activated IGF2BP3 by driving the transcription factor c-JUN, consequently modulated the m6A modification of LILRB4 mRNA and promoted immune evasion in AML. Finally, CSN5i-3 effectively disrupted the signaling pathway mediated by Jab1, thereby restoring cellular immune surveillance and halting the progression of AML. Thus, our results highlight the functional role of Jab1 in supporting AML survival and support the development of targeted therapeutic strategies.
Authors
Nan Zhang, Qian Wang, Guopeng Chen, Li Liu, Zhiying Wang, Linlu Ma, Yuxing Liang, Jinxian Wu, Xinqi Li, Xiaoyan Liu, Fuling Zhou
Abstract
The leukemia fusion gene CBFB-MYH11 requires RUNX1 for leukemogenesis, but the underlying mechanism is unclear. By in vitro studies, we found that CBFβ-SMMHC, the chimeric protein encoded by CBFB-MYH11, could enhance the binding affinity between RUNX1 and its target DNA. Increased RUNX1-DNA binding was also observed in myeloid progenitor cells from mice expressing CBFβ-SMMHC. Moreover, only CBFβ-SMMHC variants able to enhance the DNA binding affinity by RUNX1 could induce leukemia in mouse models. Marked transcriptomic changes, affecting genes associated with inflammatory response and target genes of CBFA2T3, were observed in mice expressing leukemogenic CBFβ-SMMHC variants. Finally, we show that CBFβ-SMMHC could not induce leukemia in mice with a Runx1-R188Q mutation, which reduces RUNX1 DNA binding but not affecting its interaction with CBFβ-SMMHC or its sequestration to cytoplasm by CBFβ-SMMHC. Our data suggest that, in addition to binding RUNX1 to regulate gene expression, enhancing RUNX1 binding affinity to its target DNA is an important mechanism by which CBFβ-SMMHC contributes to leukemogenesis, highlighting RUNX1–DNA interaction as a potential therapeutic target in inv(16) AML.
Authors
Tao Zhen, Yaqiang Cao, Tongyi Dou, Yun Chen, Guadalupe Lopez, Ana Catarina Menezes, Xufeng Wu, John Hammer, Jun Cheng, Lisa Garrett, Stacie Anderson, Martha Kirby, Stephen Wincovitch, Bayu Sisay, Abdel G. Elkahloun, Di Wu, Lucio H. Castilla, Wei Yang, Jiansen Jiang, Keji Zhao, Paul P. Liu
Abstract
The fetal liver is the primary site of hematopoietic stem cell (HSC) generation during embryonic development. However, the molecular mechanisms governing the transition of hematopoiesis from the fetal liver to the bone marrow (BM) remain incompletely understood. Here, we identify the mammalian Polycomb group (PcG) protein Yin Yang 1 (YY1) as a key regulator of this developmental transition. Conditional deletion of Yy1 in the hematopoietic system during fetal development results in neonatal lethality and depletion of the fetal HSC pool. YY1-deficient fetal HSCs exhibit impaired migration and fail to engraft in the adult BM, thereby losing their ability to reconstitute hematopoiesis. Transcriptomic analysis reveals that Yy1 knockout disrupts genetic networks controlling cell motility and adhesion in fetal hematopoietic stem and progenitor cells (HSPCs). Notably, YY1 does not directly bind the promoters of most dysregulated genes. Instead, it modulates chromatin accessibility at regulatory loci, orchestrating broader epigenetic programs essential for HSPC migration and adhesion. Together, these findings establish YY1 as a critical epigenetic regulator of fetal HSC function and provide a mechanistic framework to further decipher how temporal epigenomic configurations determine HSC fetal-to-adult transition during development.
Authors
Sahitya Saka, Zhanping Lu, Yinghua Wang, Peng Liu, Deependra K. Singh, Junki P. Lee, Carmen G. Palii, Tyler R. Alvarez, Anna L. F. V. Assumpção, Xiaona You, Jing Zhang, Marjorie Brand, Michael L. Atchison, Xuan Pan
Abstract
Erythropoietic protoporphyria (EPP) is a genetic disorder typically resulting from decreased ferrochelatase (FECH) activity, the last enzyme in heme biosynthesis. Patients with X-linked protoporphyria (XLPP) have an overlapping phenotype caused by increased activity of 5-aminolevulinic acid synthase 2 (ALAS2), the first enzyme in erythroid heme synthesis. In both cases, protoporphyrin IX (PPIX) accumulates in erythrocytes and secondarily in plasma and tissues. Patients develop acute phototoxicity reactions upon brief exposure to sunlight. Some also suffer from chronic liver disease, and a small fraction develop acute cholestatic liver failure. Therapeutic options are limited, and none, save hematopoietic stem cell transplantation, directly targets erythroid PPIX accumulation. Bitopertin is an investigational orally available small molecule inhibitor of the erythroid cell surface glycine transporter GLYT1. We establish the bitopertin PPIX inhibitory half-maximal effective concentration in a human erythroblast EPP model and confirm a marked reduction of PPIX in erythroblasts derived from EPP patients. We demonstrate that bitopertin also reduces erythrocyte and plasma PPIX accumulation in vivo in mouse models of both EPP and XLPP. Finally, the reduction in erythroid PPIX ameliorates liver disease in the EPP mouse model. Altogether, these data support the development of bitopertin to treat patients with EPP or XLPP.
Authors
Sarah Ducamp, Min Wu, Juan Putra, Dean R. Campagna, Yi Xiang, Vu Hong, Matthew M. Heeney, Amy K. Dickey, Rebecca K. Leaf, Mark D. Fleming, Brian MacDonald, Paul J. Schmidt
Abstract
Patients with sickle cell disease (SCD) frequently receive red blood cell (RBC) units stored near the end of their permissible storage life. To evaluate whether storage duration influences recipient metabolism, clinical chemistry and hematological parameters, we conducted a prospective, randomized, blinded trial comparing transfusions of RBC units stored for ≤10 days versus ≥30 days. Chronically transfused adults with SCD (N=24) received three consecutive outpatient transfusions with randomized-age RBCs, and blood samples from units and recipients were analyzed by metabolomics and clinical chemistry. Transfusion of short-stored units resulted in significantly higher circulating levels of 2,3-bisphosphoglycerate, an essential regulator of oxygen unloading, up to two weeks post-transfusion. Conversely, transfusions of long-stored RBCs were associated with lower hemoglobin and RBC increments, higher iron and transferrin saturation, pro-inflammatory cytokines and metabolites, oxidative stress and markers of renal dysfunction. Plasma and RBC metabolomic profiles revealed time- and storage-age-dependent alterations, particularly affecting glycolysis, purine, and sphingolipid metabolism. Transfusion of long-stored RBCs consistently worsened laboratory surrogates of oxygen delivery and RBC efficacy, and increased the circulating levels of immunomodulatory metabolites and pro-inflammatory cytokines. These findings highlight metabolic and hematologic advantages associated with transfusing fresher RBCs in adults with SCD, independent of immediate clinical outcomes.
Authors
Matthew S. Karafin, Abby L. Grier, Ross M. Fasano, Anton Ilich, David Wichlan, Ada Chang, Sonjile M. James, Hailly E. Butler, Oleg Kolupaev, Melissa C. Caughey, Daniel J Stephenson, Julie A. Reisz, Nigel S. Key, Joshua J. Field, Jane A. Little, Steven L. Spitalnik, Angelo D’Alessandro
Abstract
Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSB) that require repair through pathways such as homologous recombination, non-homologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1) is a well-known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined its impact on RS and DSB-repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activates the polymerase theta (POLQ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1-induced RS. Moreover, concurrent ATM deficiency further intensifies RS, enhances POLQ expression and heightens reliance on MMEJ mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1-overexpressed settings further exacerbates RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is, therefore, an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.
Authors
Jithma Abeykoon, Shuhei Asada, Guangli Zhu, Yuna Hirohashi, Lisa Moreau, Divya R. Iyer, Sirisha Mukkavalli, Kalindi Parmar, Gabriella Zambrano, Lige Jiang, Dongni Yi, Michelle Manske, Kimberly Gwin, Rebecca L. King, James R. Cerhan, Xiaosheng Wu, Zhenkun Lou, Geoffrey I. Shapiro, Thomas Witzig, Alan D'Andrea
Abstract
The balance of hematopoietic stem cell (HSC) self-renewal versus differentiation is essential to ensure long-term repopulation capacity while allowing response to events that require increased hematopoietic output. Proliferation and differentiation of HSCs and their progeny is controlled by the JAK/STAT pathway downstream of cytokine signaling. E3 ubiquitin ligases, like Cullin 5 (Cul5), can regulate JAK/STAT signaling by degrading signaling intermediates. Here we report that mice lacking Cul5 in hematopoietic cells (Cul5Vav-Cre) have increased numbers of HSPCs, splenomegaly, and extramedullary hematopoiesis. Differentiation in Cul5Vav-Cre mice is myeloid- and megakaryocyte-biased, resulting in leukocytosis, anemia and thrombocytosis. Cul5Vav-Cre mice increased HSC proliferation and circulation, associated with a decrease in CXCR4 surface expression. In bone marrow cells, we identified LRRC41 co-immunoprecipitated with CUL5, and vice versa, supporting that CRL5 forms a complex with LRRC41. We identified an accumulation of LRRC41 and STAT5 in Cul5Vav-Cre HSCs during IL-3 stimulation, supporting their regulation by Cul5. Whole cell proteome (WCP) analysis of HSPCs from Cul5Vav-Cre bone marrow identified upregulation of many STAT5 target genes and associated pathways. Finally, JAK1/2 inhibition with ruxolitinib normalized hematopoiesis in Cul5Vav-Cre mice. These studies demonstrate the function of Cul5 in HSC function, stem cell fate decisions, and regulation of IL-3 signaling.
Authors
Siera A. Tomishima, Dale D. Kim, Nadia Porter, Ipsita Guha, Asif A. Dar, Yohaniz Ortega-Burgos, Jennifer Roof, Hossein Fazelinia, Lynn A. Spruce, Christopher S. Thom, Robert L. Bowman, Paula M. Oliver
Abstract
BACKGROUND. Axicabtagene ciloleucel (axi-cel), anti-CD19 chimeric antigen receptor (CAR) T-cell therapy demonstrated remarkable efficacy with manageable toxicity in relapsed/refractory indolent B-cell lymphomas in the ZUMA-5 trial. METHODS. Here, we report associations of product attributes, serum biomarkers, clinical features, and tumor characteristics with outcome in 124 follicular lymphoma (FL) patients. RESULTS. In univariate and multivariate analyses, pre-treatment inflammatory markers, including TNFα and IL12p40, as well as total metabolic tumor volume (TMTV) associated with disease progression. Conversely, T-naïve-like product phenotype associated with improved outcome, particularly in high TMTV patients. These covariates improved risk stratification when combined with the FL International Prognostic Index. Post-infusion, CAR T-cell expansion associated with improved outcome, while serum inflammatory and immuno-modulatory markers, including TNFα associated with disease progression and occurrence of high-grade cytokine release syndrome or neurologic events, presenting targets to improve the therapeutic index of axi-cel in FL. Tumor gene expression profiling revealed that both type I and II IFN signaling associated with disease progression and higher expression of T cell exhaustion markers, including TIM3 and LAG3. Pre- or post-treatment CD19 expression on tumor was not associated with outcome. CONCLUSION. These findings offer insights into mechanisms of resistance and toxicity, risk stratification, and strategies for development of next generation CAR-T approaches. TRIAL REGISTRATION. ClinicalTrials.gov NCT03105336. FUNDING. Kite, a Gilead Company.
Authors
Soumya Poddar, Jiali Yan, Gayatri Tiwari, Darawan Rinchai, Justin Budka, Wangshu Zhang, Weixin Peng, Shruti Salunkhe, Madison Davis, Qinghua Song, Sara Beygi, Harry Miao, Mike Mattie, Rhine S. Shen, Caron A. Jacobson, Davide Bedognetti, Simone Filosto, Sattva S. Neelapu
Abstract
Authors
Caitlin M. Stewart, Sonya Parpart-Li, James R. White, Mitesh Patel, Oliver Artz, Michael B. Foote, Erika Gedvilaite, Michelle F. Lamendola-Essel, Drew Gerber, Rohini Bhattacharya, Justin M. Haseltine, Kety Huberman, Kelly L. Bolton, Ross L. Levine, Luis A. Diaz, Jr.
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)