TP53 mutations and TET2 deficiency cooperate to drive leukemogenesis and establish an immunosuppressive environment

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Abstract

Mutations and deletions in TP53 are associated with adverse outcomes in patients with myeloid malignancies and developing improved therapies for TP53-mutant leukemias is of urgent need. Here we identify mutations in TET2 as the most common co-occurring mutation in TP53 mutant acute myeloid leukemia (AML) patients. In mice, combined hematopoietic-specific deletion of TET2 and TP53 resulted in enhanced self-renewal compared to deletion of either gene alone. Tp53/Tet2 double knockout mice developed serially transplantable AML. Both mice and AML patients with combined TET2/TP53 alterations upregulated innate immune signaling in malignant granulocyte-monocyte progenitors (GMPs), which had leukemia-initiating capacity. A20 governs the leukemic maintenance by triggering aberrant non-canonical NF-κB signaling. Mice with Tp53/Tet2 loss had expansion of monocytic myeloid-derived suppressor cells (MDSCs), which impaired T cell proliferation and activation. Moreover, mice and AML patients with combined TP53/TET2 alterations displayed increased expression of the TIGIT ligand, CD155, on malignant cells. TIGIT blocking antibodies augmented NK cell-mediated killing of Tp53/Tet2 double-mutant AML cells, reduced leukemic burden, and prolonged survival in Tp53/Tet2 double knockout mice. These findings uncover a leukemia-promoting link between TET2 and TP53 mutations and highlight therapeutic strategies to overcome the immunosuppressive bone marrow environment in this adverse subtype of AML.

Authors

Pu Zhang, Ethan C. Whipp, Sarah J. Skuli, Mehdi Gharghabi, Caner Saygin, Steven A. Sher, Martin Carroll, Xiangyu Pan, Eric D. Eisenmann, Tzung-Huei Lai, Bonnie K. Harrington, Wing Keung Chan, Youssef Youssef, Bingyi Chen, Alex Penson, Alexander M. Lewis, Cynthia R. Castro, Nina Fox, Ali Cihan, Jean-Benoit Le Luduec, Susan DeWolf, Tierney Kauffman, Alice S. Mims, Daniel Canfield, Hannah Phillips, Katie E. Williams, Jami Shaffer, Arletta Lozanski, Tzyy-Jye Doong, Gerard Lozanski, Charlene Mao, Christopher J. Walker, James S. Blachly, Anthony F. Daniyan, Lapo Alinari, Robert A. Baiocchi, Yiping Yang, Nicole R. Grieselhuber, Moray J. Campbell, Sharyn D. Baker, Bradley W. Blaser, Omar Abdel-Wahab, Rosa Lapalombella

Proteostasis and metabolic dysfunction characterize a subset of storage-induced senescent erythrocytes targeted for post-transfusion clearance

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Abstract

Although refrigerated storage slows the metabolism of volunteer donor RBCs, which is essential in transfusion medicine, cellular aging still occurs throughout this in vitro process. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhigh) and morphologically-normal RBCs (CFSElow), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhigh RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhigh RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhigh RBC membranes. CFSEhigh RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen perfusion ex vivo. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElow RBCs resembled those of short-stored RBCs. CFSEhigh RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in these storage-induced senescent RBCs targeted for clearance.

Authors

Sandy Peltier, Mickaël Marin, Monika Dzieciatkowska, Michaël Dussiot, Micaela Kalani Roy, Johanna Bruce, Louise Leblanc, Youcef Hadjou, Sonia Georgeault, Aurélie Fricot, Camille Roussel, Daniel Stephenson, Madeleine Casimir, Abdoulaye Sissoko, François Paye, Safi Dokmak, Papa Alioune Ndour, Philippe Roingeard, Emilie-Fleur Gautier, Steven L. Spitalnik, Olivier Hermine, Pierre A. Buffet, Angelo D’Alessandro, Pascal Amireault

Restoring mitochondrial function promotes hematopoietic reconstitution from cord blood following cryopreservation-related functional decline

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Abstract

Umbilical cord blood (UCB) showcases substantial roles in hematopoietic stem cells (HSCs) transplantation and regenerative medicine. UCB is usually cryopreserved for years before use. Whether and how cryopreservation affects its function remain unclear. We constructed single-cell transcriptomic profile of CD34+ hematopoietic stem and progenitor cells (HSPCs) and mononuclear cells (MNCs) from fresh and cryopreserved UCB stored for 1-, 5-, 10-, and 19- years. Compared to fresh UCB, cryopreserved HSCs and multipotent progenitors (MPPs) exhibited more active cell cycle and lower HSC/MPP signature gene expressions. Hematopoietic reconstitution of cryopreserved HSPCs gradually decreased during the first 5 years but stabilized thereafter, aligning with the negative correlation between clinical neutrophil engraftment and cryopreservation duration of UCB. Cryopreserved HSPCs also showed reduced megakaryocyte generation. In contrast, cryopreserved natural killer (NK) cells and T cells maintained cytokine production and cytotoxic ability comparable to fresh cells. Mechanistically, cryopreserved HSPCs exhibited elevated reactive oxygen species, reduced ATP synthesis, and abnormal mitochondrial distribution, which collectively led to attenuated hematopoietic reconstitution. These effects could be ameliorated by sulforaphane. Together, we elucidated the negative impact of cryopreservation on UCB HSPCs and provided sulforaphane as a mitigation strategy, broadening the temporal window and scope for clinical applications of cryopreserved UCB.

Authors

Yaojin Huang, Xiaowei Xie, Mengyao Liu, Yawen Zhang, Junye Yang, Wenling Yang, Yu Hu, Saibing Qi, Yahui Feng, Guojun Liu, Shihong Lu, Xuemei Peng, Jinhui Ye, Shihui Ma, Jiali Sun, Lu Wang, Linping Hu, Lin Wang, Xiaofan Zhu, Hui Cheng, Zimin Sun, Junren Chen, Fang Dong, Yingchi Zhang, Tao Cheng

Megakaryocytes transfer mitochondria to bone marrow mesenchymal stromal cells to lower platelet activation

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Abstract

Newly produced platelets acquire a low activation state but whether the megakaryocyte plays a role in this outcome has not been fully uncovered. Mesenchymal stem cells (MSCs) were previously shown to promote platelet production and lower platelet activation. We found healthy megakaryocytes transfer mitochondria to MSCs mediated by Connexin 43 (Cx43) gap junctions on MSCs, which leads to platelets at a low energetic state with increased LYN activation, characteristic of resting platelets. On the contrary, MSCs have a limited ability to transfer mitochondria to megakaryocytes. Sickle cell disease (SCD) is characterized by hemolytic anemia and results in heightened platelet activation, contributing to numerous disease complications. Platelets in SCD mice and human patient samples had a heightened energetic state with increased glycolysis. MSC exposure to heme in SCD led to decreased Cx43 expression and a reduced ability to uptake mitochondria from megakaryocytes. This prevented LYN activation in platelets and contributed to increased platelet activation at steady state. Altogether, our findings demonstrate an effect of hemolysis in the microenvironment leading to increased platelet activation in SCD. These findings have the potential to inspire new therapeutic targets to relieve thrombosis-related complications of SCD and other hemolytic conditions.

Authors

Chengjie Gao, Yitian Dai, Paul A. Spezza, Paul Boasiako, Alice Tang, Giselle Rasquinha, Hui zhong, Bojing Shao, Yunfeng Liu, Patricia A. Shi, Cheryl A. Lobo, Xiuli An, Anqi Guo, William B. Mitchell, Deepa Manwani, Karina Yazdanbakhsh, Avital Mendelson

Reduced EIF6 dosage attenuates TP53 activation in models of Shwachman-Diamond syndrome

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Abstract

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency, and bony abnormalities with an increased risk of myeloid neoplasia. Almost all cases of SDS result from biallelic mutations in SBDS. SBDS interacts with EFL1 to displace EIF6 from the 60S ribosomal subunit. Released EIF6 permits the assembly of ribosomal large and small subunits in the cytoplasm. Decreased EIF6 levels due to haploinsufficiency or missense mutations which lead to decreased protein expression may provide a somatic genetic rescue and anti-leukemic effects. We observed accumulation of EIF6 protein in sbds knockout (KO) zebrafish models, confirmed in patient-derived tissues, and correlated with changes in ribosome proteins and TP53 pathways. The mechanism of action for this adaptive response is unknown. To address this, we generated an eif6 zebrafish KO line which do not survive past 10 days post fertilization. We also created two mutants with low Eif6 expression, 5-25% of the wildtype levels, that can survive until adulthood. We bred them with sbds-null strains and analyzed their phenotype and biochemical properties. Low Eif6 levels reduced Tp53 pathway activation but did not rescue neutropenia in Sbds-deficient zebrafish. Further studies elucidating the interplay between SBDS, EIF6, TP53, and cellular stress responses offer promising insights into SDS pathogenesis, somatic genetic rescue, and therapeutic strategies.

Authors

Usua Oyarbide, Valentino Bezzerri, Morgan Staton, Christian Boni, Arish Shah, Marco Cipolli, Eliezer Calo, Seth J. Corey

HoxBlinc lncRNA reprograms CTCF-independent TADs to drive leukemic transcription and HSC dysregulation in NUP98-rearranged leukemia

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Abstract

Although nucleoporin 98 (NUP98) fusion oncogenes often drive aggressive pediatric leukemia by altering chromatin structure and expression of HOX genes, underlying mechanisms remain elusive. Here, we report that a Hoxb-associated lncRNA HoxBlinc was aberrantly activated in NUP98-PHF23 fusion-driven leukemias. HoxBlinc chromatin occupancies led to elevated MLL1 recruitment and aberrant homeotic topologically associated domains (TADs) that enhanced chromatin accessibilities and activated homeotic/hematopoietic oncogenes. HoxBlinc-depletion in NUP98 fusion-driven leukemia impaired HoxBlinc binding, TAD integrity, MLL1 recruitment, and MLL1-driven chromatin signature within HoxBlinc-defined TADs in a CTCF-independent manner, leading to inhibited homeotic/leukemic oncogenes that mitigated NUP98 fusion-driven leukemogenesis in xenografted mouse models. Mechanistically, HoxBlinc overexpression in mouse hematopoietic compartment induced leukemias resembling those in NUP98-PHF23 knock-in mice via enhancing HoxBlinc chromatin binding, TAD formation, and Hox gene aberration leading to expansion of hematopoietic stem and progenitor cell (HSPC) and myeloid/lymphoid subpopulations. Thus, our studies reveal a CTCF-independent role of HoxBlinc in leukemic TAD organization and oncogene regulatory networks.

Authors

Karina Hamamoto, Ganqian Zhu, Qian Lai, Julia Lesperance, Huacheng Luo, Ying Li, Nupur Nigam, Arati Sharma, Feng-Chun Yang, David Claxton, Yi Qiu, Peter D. Aplan, Mingjiang Xu, Suming Huang

Human Oncostatin M deficiency underlies an inherited severe bone marrow failure syndrome

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Abstract

Oncostatin M (OSM) is a cytokine with the unique ability to interact with both the OSM receptor (OSMR) and the leukemia inhibitory factor receptor (LIFR). On the other hand, OSMR interacts with IL31RA to form the interleukin-31 receptor. This intricate network of cytokines and receptors makes it difficult to understand the specific function of OSM. While monoallelic loss-of-function (LoF) mutations in OSMR underlie autosomal dominant familial primary localized cutaneous amyloidosis, the in vivo consequences of human OSM deficiency have never been reported so far. Here, we identified three young individuals from a consanguineous family presenting with inherited severe bone marrow failure syndromes (IBMFS) characterized by profound anemia, thrombocytopenia, and neutropenia. Genetic analysis revealed a homozygous one base-pair insertion in the sequence of OSM associated with the disease. Structural and functional analyses showed that this variant causes a frameshift that replaces the C-terminal portion of OSM, which contains the FxxK motif that interacts with both OSMR and LIFR, with a neopeptide. The lack of detection and signaling of the mutant OSM suggests a LoF mutation. Analysis of zebrafish models further supported the role of the OSM/OSMR signaling in erythroid progenitor proliferation and neutrophil differentiation. Our study provides the previously uncharacterized and unexpectedly limited in vivo consequence of OSM deficiency in humans.

Authors

Alexandrine Garrigue, Laëtitia Kermasson, Sandrine Susini, Ingrid Fert, Christopher B. Mahony, Hanem Sadek, Sonia Luce, Myriam Chouteau, Marina Cavazzana, Emmanuelle Six, Marie-Caroline Le Bousse-Kerdilès, Adrienne Anginot, Jean-Baptiste Souraud, Valérie Cormier-Daire, Marjolaine Willems, Anne Sirvent, Jennifer Russello, Isabelle Callebaut, Isabelle André, Julien Y. Bertrand, Chantal Lagresle-Peyrou, Patrick Revy

Loss of Cpt1a results in elevated glucose-fueled mitochondrial oxidative phosphorylation and defective hematopoietic stem cells

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Abstract

Hematopoietic stem cells (HSCs) rely on self-renewal to sustain stem cell potential and undergo differentiation to generate mature blood cells. Mitochondrial fatty acid β-oxidation (FAO) is essential for HSC maintenance. However, the role of Carnitine palmitoyl transferase 1a (CPT1A), a key enzyme in FAO, remains unclear in HSCs. Using a Cpt1a hematopoietic specific conditional knock-out (Cpt1aΔ/Δ) mouse model, we found that loss of Cpt1a leads to HSC defects, including loss of HSC quiescence and self-renewal, and increased differentiation. Mechanistically, we find that loss of Cpt1a results in elevated levels of mitochondrial respiratory chain complex components and their activities, as well as increased ATP production, and accumulation of mitochondrial reactive oxygen species (mitoROS) in HSCs. Taken together, this suggests hyperactivation of mitochondria and metabolic rewiring via upregulated glucose-fueled oxidative phosphorylation (OXPHOS). In summary, our findings demonstrate a novel role for Cpt1a in HSC maintenance and provide insight into the regulation of mitochondrial metabolism via control of the balance between FAO and glucose-fueled OXPHOS.

Authors

Jue Li, Jie Bai, Vincent T. Pham, Michihiro Hashimoto, Maiko Sezaki, Qili Shi, Qiushi Jin, Chenhui He, Amy Armstrong, Tian Li, Mingzhe Pan, Shujun Liu, Yu Luan, Hui Zeng, Paul R. Andreassen, Gang Huang

Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells

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Abstract

Ecotropic viral integration site 1 (EVI1/MECOM) is frequently upregulated in myeloid malignancies. Here, we present an Evi1-transgenic mouse model with inducible expression in hematopoietic stem/progenitor cells (HSPCs). Upon induction of Evi1 expression, mice displayed anemia, thrombocytopenia, lymphopenia, and erythroid and megakaryocyte dysplasia with a significant expansion of committed myeloid progenitor cells, resembling human myelodysplastic syndrome/myeloproliferative neoplasm–like (MDS/MPN–like) disease. Evi1 overexpression prompted HSPCs to exit quiescence and accelerated their proliferation, leading to expansion of committed myeloid progenitors while inhibiting lymphopoiesis. Analysis of global gene expression and Evi1 binding site profiling in HSPCs revealed that Evi1 directly upregulated lysine demethylase 6b (Kdm6b). Subsequently, Kdm6b-mediated H3K27me3 demethylation resulted in activation of various genes, including Laptm4b. Interestingly, KDM6B and LAPTM4B are positively correlated with EVI1 expression in patients with MDS. The EVI1/KDM6B/H3K27me3/LAPTM4B signaling pathway was also identified in EVI1hi human leukemia cell lines. We found that hyperactivation of the LAPTM4B-driven mTOR pathway was crucial for the growth of EVI1hi leukemia cells. Knockdown of Laptm4b partially rescued Evi1-induced abnormal hematopoiesis in vivo. Thus, our study establishes a mouse model to investigate EVI1hi myeloid malignancies, demonstrating the significance of the EVI1-mediated KDM6B/H3K27me3/LAPTM4B signaling axis in their maintenance.

Authors

Qiong Wu, Chunjie Yu, Fang Yu, Yiran Guo, Yue Sheng, Liping Li, Yafang Li, Yutao Zhang, Chao Hu, Jue Wang, Tong-chuan He, Yong Huang, Hongyu Ni, Zhiguang Huo, Wenshu Wu, Gang Greg Wang, Jianxin Lyu, Zhijian Qian

IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells

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Abstract

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T-cell receptor (TCR) signaling, while its role in CAR signaling is unknown. By electroporation of clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.

Authors

Zheng Fu, Zineng Huang, Hao Xu, Qingbai Liu, Jing Li, Keqing Song, Yating Deng, Yujia Tao, Huifang Zhang, Peilong Wang, Heng Li, Yue Sheng, Aijun Zhou, Lianbin Han, Yan Fu, Chen-Zhi Wang, Saurav Kumar Choudhary, Kaixiong Ye, Gianluca Veggiani, Zhihong Li, Avery August, Weishan Huang, Qiang Shan, Hongling Peng