Hematology
Abstract
BACKGROUND. Predicting Immune-effector Cell Associated Neurotoxicity Syndrome (ICANS) in patients infused with Chimeric Antigen Receptor T cells (CAR-T) is still a conundrum. This complication, thought to be consequent to CAR-T cell activation, arises a few days after infusion, when circulating CAR-T cells are scarce and specific CAR-T cell-derived biomarkers are lacking. METHODS. Human CD19.CAR-T cells were generated to gain insight into CAR+ extracellular vesicle (CAR+EV) release upon target engagement. A prospective cohort of 100 B-cell lymphoma patients infused with approved CD19.CAR-T cell products (axi-cel, brexu-cel and tisa-cel) was assessed for plasma CAR+EVs as potential biomarkers of in vivo CD19.CAR-T cell activation and predictors of ICANS. Human induced pluripotent stem cells (iPSCs)-derived neural cells were used as a model for CAR+EV-induced neurotoxicity. RESULTS. In vitro, exosome-like CAR+EVs were released by CD19.CAR-T cells upon target engagement. In vivo, CAR+EVs were detectable as early as 1 hour in the plasma of patients. A concentration > 132.8 CAR+EVs/μl at hour +1 or > 224.5 CAR+EVs/μl at day +1 predicted ICANS in advance of 4 days, with a sensitivity up to 96.55% and a specificity up to 80.36%, outperforming other potential ICANS predictors. Enolase 2 (ENO2+) nanoparticles were released by iPSCs-derived neural cells upon CAR+EVs exposure and were increased in the plasma of ICANS patients. CONCLUSIONS. These results convey that plasma CAR+EVs are an immediate signal of CD19.CAR-T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis. TRIAL REGISTRATION. NCT04892433, NCT05807789.
Authors
Gianluca Storci, Francesco De Felice, Francesca Ricci, Spartaco Santi, Daria Messelodi, Salvatore Nicola Bertuccio, Noemi Laprovitera, Michele Dicataldo, Lucrezia Rossini, Serena De Matteis, Beatrice Casadei, Francesca Vaglio, Margherita Ursi, Francesco Barbato, Marcello Roberto, Maria Guarino, Gian Maria Asioli, Mario Arpinati, Pietro Cortelli, Enrico Maffini, Enrica Tomassini, Marta Tassoni, Carola Cavallo, Francesco Iannotta, Maria Naddeo, Pier Luigi Tazzari, Elisa Dan, Cinzia Pellegrini, Serafina Guadagnuolo, Matteo Carella, Barbara Sinigaglia, Chiara Pirazzini, Caterina Severi, Paolo Garagnani, Katarzyna Malgorzata Kwiatkowska, Manuela Ferracin, Pier Luigi Zinzani, Massimiliano Bonafè, Francesca Bonifazi
Abstract
Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.
Authors
Xiaolei Liu, Sudhish A. Devadiga, Robert F. Stanley, Ryan M. Morrow, Kevin A. Janssen, Mathieu Quesnel-Vallières, Oz Pomp, Adam A. Moverley, Chenchen Li, Nicolas Skuli, Martin P. Carroll, Jian Huang, Douglas C. Wallace, Kristen W. Lynch, Omar Abdel-Wahab, Peter S. Klein
Abstract
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor–ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.
Authors
Tobias Suske, Helena Sorger, Gabriele Manhart, Frank Ruge, Nicole Prutsch, Mark W. Zimmerman, Thomas Eder, Diaaeldin I. Abdallah, Barbara Maurer, Christina Wagner, Susann Schönefeldt, Katrin Spirk, Alexander Pichler, Tea Pemovska, Carmen Schweicker, Daniel Pölöske, Emina Hubanic, Dennis Jungherz, Tony Andreas Müller, Myint Myat Khine Aung, Anna Orlova, Ha Thi Thanh Pham, Kerstin Zimmel, Thomas Krausgruber, Christoph Bock, Mathias Müller, Maik Dahlhoff, Auke Boersma, Thomas Rülicke, Roman Fleck, Elvin Dominic de Araujo, Patrick Thomas Gunning, Tero Aittokallio, Satu Mustjoki, Takaomi Sanda, Sylvia Hartmann, Florian Grebien, Gregor Hoermann, Torsten Haferlach, Philipp Bernhard Staber, Heidi Anne Neubauer, Alfred Thomas Look, Marco Herling, Richard Moriggl
Abstract
Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable, however designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated, drug product for targeting CD33+ tumors called dimerization agent regulated immunoreceptor complex (DARIC33). T cell products demonstrated target specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following re-exposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for OFF-ON state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase 1 DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and anti-tumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.
Authors
Jacob Appelbaum, April E. Price, Kaori Oda, Joy Zhang, Wai-Hang Leung, Giacomo Tampella, Dong Xia, Pauline P.L. So, Sarah K. Hilton, Claudya Evandy, Semanti Sarkar, Unja Martin, Anne-Rachel Krostag, Marissa Leonardi, Daniel E. Zak, Rachael Logan, Paula Lewis, Secil Franke-Welch, Njabulo Ngwenyama, Michael Fitzgerald, Niklas Tulberg, Stephanie Rawlings-Rhea, Rebecca A. Gardner, Kyle Jones, Angelica Sanabria, William Crago, John Timmer, Andrew Hollands, Brendan Eckelman, Sanela Bilic, Jim Woodworth, Adam Lamble, Philip D. Gregory, Jordan Jarjour, Mark Pogson, Joshua A. Gustafson, Alexander Astrakhan, Michael C. Jensen
Abstract
Chromosomal instability is a prominent biological feature of Myelodysplastic Syndromes (MDS), with over 50% of MDS patients harboring chromosomal abnormalities or a complex karyotype. Despite this observation, the mechanisms underlying mitotic and chromosomal defects in MDS remain elusive. In this study, we identified a ectopic expression of transcription factor ONECUT3, associated with complex karyotypes and poorer survival outcomes in MDS. ONECUT3-overexpressing cell models exhibited enrichment of several notable pathways, including signatures of sister chromosome exchange separation and mitotic nuclear division with the upregulation of INCENP and CDCA8 genes. Notably, dysregulation of Chromosome Passenger Complex (CPC) accumulation besides the cell equator and midbody during mitotic phases consequently caused cytokinesis failure and defective chromosome segregation. Mechanistically, the Homeobox (HOX) domain of ONECUT3, serving as the DNA binding domain, occupied the unique genomic regions of INCENP and CDCA8, and transcriptionally activated these two genes. A novel lead compound C5484617, was identified that functionally targeted the HOX domain of ONECUT3 inhibiting its transcriptional activity on downstream genes, and synergistically resensitized MDS cells to hypomethylating agents. This study revealed that ONECUT3 promoted chromosomal instability by transcriptional activation of INCENP and CDCA8, suggesting potential prognosis and therapeutic roles for targeting high-risk MDS patients with complex karyotype.
Authors
Yingwan Luo, Xiaomin Feng, Wei Lang, Weihong Xu, Wei Wang, Chen Mei, Li Ye, Shuanghong Zhu, Lu Wang, Xinping Zhou, Huimin Zeng, Liya Ma, Yanling Ren, Jie Jin, Rongzhen Xu, Gang Huang, Hongyan Tong
Abstract
Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates indicating activation of platelets from essential thrombocythemia (ET) patients. Single cell RNA-seq analysis of primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR and oxidative phosphorylation (OXPHOS) in ET patient platelets. These observations were validated via proteomic profiling. Platelet metabolomics revealed distinct metabolic phenotypes consisting of elevated ATP generation, accompanied by increases in the levels of multiple intermediates of the tricarboxylic acid (TCA) cycle, but lower alpha-ketoglutarate (α-KG) in MPN patients. Inhibition of PI3K/AKT/mTOR signaling significantly reduced metabolic responses and hyperreactivity in MPN patient platelets, while α-KG supplementation markedly reduced oxygen consumption and ATP generation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F mice with α-KG significantly reduced platelet activation responses. Oral α-KG supplementation of Jak2 V617F mice decreased splenomegaly and reduced hematocrit, monocyte and platelet counts. Finally, α-KG incubation significantly decreased proinflammatory cytokine secretion from MPN CD14+ monocytes. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling, contributing to platelet hyperreactivity in MPN patients.
Authors
Fan He, Angelo B.A. Laranjeira, Tim Kong, Shuyang Lin, Katrina J. Ashworth, Alice Liu, Nina M. Lasky, Daniel A.C. Fisher, Maggie J. Cox, Mary C. Fulbright, Lilian A. Antunes Heck, LaYow C. Yu, Molly Brakhane, Bei Gao, Stephen M. Sykes, Angelo D’Alessandro, Jorge A. Di Paola, Stephen T. Oh
Abstract
Gestational diabetes is a common medical complication of pregnancy that is associated with adverse perinatal outcomes and an increased risk of metabolic diseases and atherosclerosis in adult offspring. The mechanisms responsible for this delayed pathological transmission remain unknown. In mouse models, we found that the development of atherosclerosis in adult offspring born to diabetic pregnancy can be in part linked to hematopoietic alterations. Although they do not show any gross metabolic disruptions, the adult offspring maintain hematopoietic features associated with diabetes, indicating the acquisition of a lasting diabetic hematopoietic memory. We show that the induction of this hematopoietic memory during gestation relies on the activity of the AGER pattern recognition receptor and the NLRP3 inflammasome, which leads to increased placental inflammation. In adult offspring, we find that this memory is associated with DNMT1 upregulation and epigenetic changes in hematopoietic progenitors. Altogether, our results demonstrate that the hematopoietic system can acquire a lasting memory of gestational diabetes, and that this memory constitutes a new pathway connecting gestational health to adult pathologies.
Authors
Vinothini Govindarajah, Masahide Sakabe, Samantha Good, Michael Solomon, Ashok Arasu, Nong Chen, Xuan Zhang, H. Leighton Grimes, Ady Kendler, Mei Xin, Damien Reynaud
Abstract
ASXL1 mutation frequently occurs in all forms of myeloid malignancies and is associated with aggressive disease and poor prognosis. ASXL1 recruits Polycomb Repressive Complex 2 (PRC2) to specific gene loci to repress transcription through tri-methylation of histone H3 on lysine 27 (H3K27me3). ASXL1 alterations reduce H3K27me3 levels, which results in leukemogenic gene expression and the development of myeloid malignancies. Standard therapies for myeloid malignancies have limited efficacy when mutated ASXL1 is present. We discovered up-regulation of lysine demethylase 6B (KDM6B), a demethylase for H3K27me3, in ASXL1-mutant leukemic cells, which further reduces H3K27me3 levels and facilitates myeloid transformation. Here, we demonstrated that heterozygous deletion of Kdm6b restored H3K27me3 levels and normalized dysregulated gene expression in Asxl1Y588XTg hematopoietic stem/progenitor cells (HSPCs). Furthermore, heterozygous deletion of Kdm6b decreased the HSPC pool, restored their self-renewal capacity, prevented biased myeloid differentiation, and abrogated progression to myeloid malignancies in Asxl1Y588XTg mice. Importantly, administration of GSK-J4, a KDM6B inhibitor, not only restored H3K27me3 levels but also reduced the disease burden in NSG mice xenografted with human ASXL1 mutant leukemic cells in vivo. This preclinical finding provides compelling evidence that targeting KDM6B may be a therapeutic strategy for myeloid malignancies with ASXL1 mutations.
Authors
Guo Ge, Peng Zhang, Pinpin Sui, Shi Chen, Hui Yang, Ying Guo, Ivan P. Rubalcava, Asra Noor, Caroline R. Delma, Joel Agosto-Peña, Hui Geng, Edward A. Medina, Ying Liang, Stephen D. Nimer, Ruben Mesa, Omar Abdel-Wahab, Mingjiang Xu, Feng-Chun Yang
Abstract
Authors
Feng Pan, Jolanda Sarno, Johan Jeong, Xin Yang, Astraea Jager, Tanja A. Gruber, Kara L. Davis, Michael L. Cleary
Abstract
We have previously demonstrated that cystatin E/M (CST6), elevated in a subset of multiple myeloma (MM) patients lacking osteolytic lesions (OL), suppresses MM bone disease by blocking osteoclast differentiation and function. CST6 is a secreted type 2 cystatin, a cysteine protease inhibitor that regulates lysosomal cysteine proteases and the asparaginyl endopeptidase legumain. We have developed B cell maturation antigen (BCMA)-CST6-chimeric antigen receptor (CAR) T cells, which lyse MM cells and release CST6 proteins. Our in vitro studies show that these CAR-T cells suppress differentiation and formation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. Using xenografted MM mice, bioluminescence images show that both BCMA-CAR-T and BCMA-CST6-CAR-T cells inhibit MM growth to a similar extent. Reconstructed micro-computed tomography (µCT) images reveal that BCMA-CST6-CAR-T cells, but not BCMA-CAR-T cells, prevent MM-induced bone damage and decrease osteoclast numbers. Our results provide a CAR-T strategy that targets tumor cells directly and delivers an inhibitor of bone resorption.
Authors
Fumou Sun, Yan Cheng, Jin-Ran Chen, Visanu Wanchai, David E. Mery, Hongwei Xu, Dongzheng Gai, Samer Al Hadidi, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr., Fenghuang Zhan
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ISSN: 0021-9738 (print), 1558-8238 (online)