p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1−/−p53−/− mouse model. However, the mechanisms triggering p53 activation in Mysm1−/− HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1−/− phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1−/− HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1−/− hematopoietic dysfunction. Specifically, Mysm1−/−Puma−/− mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1−/−Puma−/− MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1−/−Puma−/− MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1−/−hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.

Key Points Developmental defects and impairment in lymphopoiesis in Mysm1-knockout mice are the result of p53 activation. Loss of hematopoietic stem cell function and bone marrow failure in Mysm1-knockout mice are the result of p53 activation.

Acute myeloid leukemia (AML) bears heterogeneous cells that can consequently offset killing by single-CAR-based therapy, which results in disease relapse. Leukemic stem cells (LSCs) associated with CD123 expression comprise a rare population that also plays an important role in disease progression and relapse. Here, we report on the robust anti-tumor activity of a compound CAR (cCAR) T-cell possessing discrete scFv domains targeting two different AML antigens, CD123, and CD33, simultaneously. We determined that the resulting cCAR T-cells possessed consistent, potent, and directed cytotoxicity against each target antigen population. Using four leukemia mouse models, we found superior in vivo survival after cCAR treatment. We also designed an alemtuzumab safety-switch that allowed for rapid cCAR therapy termination in vivo. These findings indicate that targeting both CD123 and CD33 on AML cells may be an effective strategy for eliminating both AML bulk disease and LSCs, and potentially prevent relapse due to antigen escape or LSC persistence.

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

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MYSM1 is a chromatin-interacting deubiquitinase recently shown to be essential for hematopoietic stem cell (HSC) function and normal progression of hematopoiesis in both mice and humans. However, it remains unknown whether the loss of function in Mysm1-deficient HSCs is due to the essential role of MYSM1 in establishing the HSC pool during development or due to a continuous requirement for MYSM1 in adult HSCs. In this study we, for the first time, address these questions first, by performing a detailed analysis of hematopoiesis in the fetal livers of Mysm1-knockout mice, and second, by assessing the effects of an inducible Mysm1 ablation on adult HSC functions. Our data indicate that MYSM1 is essential for normal HSC function and progression of hematopoiesis in the fetal liver. Furthermore, the inducible knockout model demonstrates a continuous requirement for MYSM1 to maintain HSC functions and antagonize p53 activation in adult bone marrow. These studies advance our understanding of the role of MYSM1 in HSC biology, and provide new insights into the human hematopoietic failure syndrome resulting from MYSM1 deficiency.

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Acute myeloid leukemia (AML) is an aggressive malignancy lacking targeted therapy due to shared molecular and transcriptional circuits as well as phenotypic markers with normal hematopoietic stem cells (HSCs). Identifying leukemia specific markers expressed on AML or AML subtypes for therapeutic targeting is of exquisite clinical value. Here we show that CD4, a T lymphocytes membrane glycoprotein that interacts with major histocompatibility complex class II antigens and is also expressed in certain AML subsets but not on HSCs is a proper target for genetically engineered chimeric antigen receptor T cells (CAR-T cells). Treatment with CD4 redirected CAR-T cell (CD4CAR) specifically eliminated CD4-expressing AML cell lines in vitro and exhibited a potent anti-leukemic effect in a systemic AML murine model in vivo . We also utilized natural killers as another vehicle for CAR engineered cells and this strategy similarly and robustly eliminated CD4- expressing AML cells in vitro and had a potent in vivo anti-leukemic effect and was noted to have shorter in vivo persistence. Our data offer a proof of concept for immunotherapeutic targeting of CD4 as a strategy to treat CD4 expressing refractory AML as a bridge to stem cell transplant (SCT) in a first in human clinical trial.