Hematology
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
Abstract
We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have extremely poor prognosis, and most of these TP53 mutations are missense mutations. Here, we report that in contrast to mixed AML and T-cell malignancy developed in NrasG12D/+; p53-/- (NP-/-) mice, NrasG12D/+; p53R172H/+ (NPmut) mice rapidly developed an inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and largely normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergize to establish an NPmut-AML transcriptome distinct from that of NP-/- cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated expression of inflammatory genes, including those linked to NFκB signaling. NFκB was also upregulated in human NRAS;TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NFκB inhibition in vitro. The proteasome inhibitor bortezomib stabilized NFκB inhibitory protein IκBα reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergizes with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.
Authors
Adhithi Rajagopalan, Yubin Feng, Meher B. Gayatri, Erik A. Ranheim, Taylor Klungness, Daniel R. Matson, Moon Hee Lee, Mabel Minji Jung, Yun Zhou, Xin Gao, Kalyan V.G. Nadiminti, David T. Yang, Vu L. Tran, Eric Padron, Shigeki Miyamoto, Emery H. Bresnick, Jing Zhang
Abstract
The BCL-2 inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B-cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL-2 mutations have been described, this likely only explains a subset of resistant cases. Using two complementary functional precision medicine techniques -- BH3-profiling and high throughput-kinase activity mapping -- we found that hyperphosphorylation of BCL-2 family proteins, including anti-apoptotic MCL-1 and BCL-2 and pro-apoptotic BAD and BAX, underlies functional mechanisms of both intrinsic and acquired resistance of venetoclax in CLL and DLBCL. Additionally, we provide evidence that anti-apoptotic BCL-2 family protein phosphorylation alters the apoptotic protein interactome, thereby changing the profile of functional dependence on these pro-survival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs re-wired these dependences, thus restoring sensitivity to venetoclax in a panel of venetoclax resistant lymphoid cell lines, resistant mouse model, and paired patient samples pre-venetoclax and at time of progression.
Authors
Stephen Jun Fei Chong, Fen Zhu, Olga Dashevsky, Rin Mizuno, Jolin X.H. Lai, Liam Hackett, Christine E. Ryan, Mary C. Collins, J. Bryan Iorgulescu, Romain Guièze, Johany Penailillo, Ruben Carrasco, Yeonjoo C. Hwang, Denise P. Muñoz, Mehdi Bouhaddou, Yaw Chyn Lim, Catherine J. Wu, John N. Allan, Richard R. Furman, Boon Cher Goh, Shazib Pervaiz, Jean-Philippe Coppé, Constantine S. Mitsiades, Matthew S. Davids
Abstract
Red blood cells (RBCs) mediate cardioprotection via nitric oxide–like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase–dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.
Authors
Jiangning Yang, Michaela L. Sundqvist, Xiaowei Zheng, Tong Jiao, Aida Collado, Yahor Tratsiakovich, Ali Mahdi, John Tengbom, Evanthia Mergia, Sergiu-Bogdan Catrina, Zhichao Zhou, Mattias Carlström, Takaaki Akaike, Miriam M. Cortese-Krott, Eddie Weitzberg, Jon O. Lundberg, John Pernow
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)