SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia.

Acute myeloid leukemias (AML) are severe hematomalignancies with dismal prognosis. The post-translational modification SUMOylation plays key roles in leukemogenesis and AML response to therapies. Here, we show that TAK-981 (subasumstat), a first-in-class SUMOylation inhibitor, is endowed with potent anti-leukemic activity in various preclinical models of AML. TAK-981 targets AML cell lines and patient blast cells in vitro and in vivo in xenografted mice with minimal toxicity on normal hematopoietic cells. Moreover, it synergizes with 5-azacytidine (AZA), a DNA-hypomethylating agent now used in combination with the BCL-2 inhibitor venetoclax to treat AML patients unfit for standard chemotherapies. Interestingly, TAK-981+AZA combination shows higher anti-leukemic activity than AZA+venetoclax combination both in vitro and in vivo, at least in the models tested. Mechanistically, TAK-981 potentiates the transcriptional reprogramming induced by AZA, promoting apoptosis, alteration of the cell cycle and differentiation of the leukemic cells. In addition, TAK-981+AZA treatment induces many genes linked to inflammation and immune response pathways. In particular, this leads to the secretion of type-I interferon by AML cells. Finally, TAK-981+AZA induces the expression of natural killer-activating ligands (MICA/B) and adhesion proteins (ICAM-1) at the surface of AML cells. Consistently, TAK-981+AZA-treated AML cells activate natural killer cells and increase their cytotoxic activity. Targeting SUMOylation with TAK-981 may thus be a promising strategy to both sensitize AML cells to AZA and reduce their immune-escape capacities.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

Multiple Fra-1-bound enhancers showing different molecular and functional features can cooperate to repress gene transcription.

BACKGROUND: How transcription factors (TFs) down-regulate gene expression remains ill-understood, especially when they bind to multiple enhancers contacting the same gene promoter. In particular, it is not known whether they exert similar or significantly different molecular effects at these enhancers. RESULTS: To address this issue, we used a particularly well-suited study model consisting of the down-regulation of the TGFB2 gene by the TF Fra-1 in Fra-1-overexpressing cancer cells, as Fra-1 binds to multiple enhancers interacting with the TGFB2 promoter. We show that Fra-1 does not repress TGFB2 transcription via reducing RNA Pol II recruitment at the gene promoter but by decreasing the formation of its transcription-initiating form. This is associated with complex long-range chromatin interactions implicating multiple molecularly and functionally heterogeneous Fra-1-bound transcriptional enhancers distal to the TGFB2 transcriptional start site. In particular, the latter display differential requirements upon the presence and the activity of the lysine acetyltransferase p300/CBP. Furthermore, the final transcriptional output of the TGFB2 gene seems to depend on a balance between the positive and negative effects of Fra-1 at these enhancers. CONCLUSION: Our work unveils complex molecular mechanisms underlying the repressive actions of Fra-1 on TGFB2 gene expression. This has consequences for our general understanding of the functioning of the ubiquitous transcriptional complex AP-1, of which Fra-1 is the most documented component for prooncogenic activities. In addition, it raises the general question of the heterogeneity of the molecular functions of TFs binding to different enhancers regulating the same gene.

New roles for AP-1/JUNB in cell cycle control and tumorigenic cell invasion via regulation of cyclin E1 and TGF-ß2.

BACKGROUND: JUNB transcription factor contributes to the formation of the ubiquitous transcriptional complex AP-1 involved in the control of many physiological and disease-associated functions. The roles of JUNB in the control of cell division and tumorigenic processes are acknowledged but still unclear. RESULTS: Here, we report the results of combined transcriptomic, genomic, and functional studies showing that JUNB promotes cell cycle progression via induction of cyclin E1 and repression of transforming growth factor (TGF)-ß2 genes. We also show that high levels of JUNB switch the response of TGF-ß2 stimulation from an antiproliferative to a pro-invasive one, induce endogenous TGF-ß2 production by promoting TGF-ß2 mRNA translation, and enhance tumor growth and metastasis in mice. Moreover, tumor genomic data indicate that JUNB amplification associates with poor prognosis in breast and ovarian cancer patients. CONCLUSIONS: Our results reveal novel functions for JUNB in cell proliferation and tumor aggressiveness through regulation of cyclin E1 and TGF-ß2 expression, which might be exploited for cancer prognosis and therapy.

The NADPH oxidase NOX2 is a marker of adverse prognosis involved in chemoresistance of acute myeloid leukemias.

Resistance to chemotherapeutic drugs is a major cause of treatment failure in acute myeloid leukemias (AML). To better characterize the mechanisms of chemoresistance, we first identified genes whose expression is dysregulated in AML cells resistant to daunorubicin or cytarabine, the main drugs used for induction therapy. The genes found to be activated are mostly linked to immune signaling and inflammation. Among them, we identified a strong upregulation of the NOX2 NAPDH oxidase subunit genes (CYBB, CYBA, NCF1, NCF2, NCF4 and RAC2). The ensuing increase in NADPH oxidase expression and production of reactive oxygen species, which is particularly strong in daunorubicin-resistant cells, participates in the acquisition and/or maintenance of resistance to daunorubicin. Gp91phox (CYBB-encoded Nox2 catalytic subunit), was found to be more expressed and active in leukemic cells from patients with the French-American-British (FAB) M4/M5 subtypes of AML than in those from patients with the FAB M0-M2 ones. Moreover, its expression was increased at the surface of patients' chemotherapy-resistant AML cells. Finally, using a gene expression based score we demonstrated that high expression of NOX2 subunit genes is a marker of adverse prognosis in AML patients. The prognostic NOX score we defined is independent of the cytogenetic-based risk classification, FAB subtype, FLT3/NPM1 mutational status and age.

SUMO and Transcriptional Regulation: The Lessons of Large-Scale Proteomic, Modifomic and Genomic Studies.

One major role of the eukaryotic peptidic post-translational modifier SUMO in the cell is transcriptional control. This occurs via modification of virtually all classes of transcriptional actors, which include transcription factors, transcriptional coregulators, diverse chromatin components, as well as Pol I-, Pol II- and Pol III transcriptional machineries and their regulators. For many years, the role of SUMOylation has essentially been studied on individual proteins, or small groups of proteins, principally dealing with Pol II-mediated transcription. This provided only a fragmentary view of how SUMOylation controls transcription. The recent advent of large-scale proteomic, modifomic and genomic studies has however considerably refined our perception of the part played by SUMO in gene expression control. We review here these developments and the new concepts they are at the origin of, together with the limitations of our knowledge. How they illuminate the SUMO-dependent transcriptional mechanisms that have been characterized thus far and how they impact our view of SUMO-dependent chromatin organization are also considered.

Fra-1 regulates its target genes via binding to remote enhancers without exerting major control on chromatin architecture in triple negative breast cancers.

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.

Ubiquitin and SUMO conjugation as biomarkers of acute myeloid leukemias response to chemotherapies.

Ubiquitin and the ubiquitin-like SUMO are covalently conjugated to thousands of proteins to modulate their function and fate. Many of the enzymes involved in their conjugation are dysregulated in cancers and involved in cancer cell response to therapies. We describe here the identification of biomarkers of the activity of these enzymes and their use to predict acute myeloid leukemias (AML) response to standard chemotherapy (daunorubicin-DNR and cytarabine-Ara-C). We compared the ability of extracts from chemosensitive and chemoresistant AML cells to conjugate ubiquitin or SUMO-1 on 9,000 proteins spotted on protein arrays. We identified 122 proteins whose conjugation by these posttranslational modifiers marks AML resistance to DNR and/or Ara-C. Based on this signature, we defined a statistical score predicting AML patient response to standard chemotherapy. We finally developed a miniaturized assay allowing for easy assessment of modification levels of the selected biomarkers and validated it in patient cell extracts. Thus, our work identifies a new type of ubiquitin-based biomarkers that could be used to predict cancer patient response to treatments.

Ubiquitin, SUMO, and Nedd8 as Therapeutic Targets in Cancer.

Ubiquitin defines a family of approximately 20 peptidic posttranslational modifiers collectively called the Ubiquitin-like (UbLs). They are conjugated to thousands of proteins, modifying their function and fate in many ways. Dysregulation of these modifications has been implicated in a variety of pathologies, in particular cancer. Ubiquitin, SUMO (-1 to -3), and Nedd8 are the best-characterized UbLs. They have been involved in the regulation of the activity and/or the stability of diverse components of various oncogenic or tumor suppressor pathways. Moreover, the dysregulation of enzymes responsible for their conjugation/deconjugation has also been associated with tumorigenesis and cancer resistance to therapies. The UbL system therefore constitutes an attractive target for developing novel anticancer therapeutic strategies. Here, we review the roles and dysregulations of Ubiquitin, SUMO, and Nedd8 pathways in tumorigenesis, as well as recent advances in the identification of small molecules targeting their conjugating machineries for potential application in the fight against cancer.

The SUMO Pathway in Hematomalignancies and Their Response to Therapies.

SUMO (Small Ubiquitin-related MOdifier) is a post-translational modifier of the ubiquitin family controlling the function and fate of thousands of proteins. SUMOylation is deregulated in various hematological malignancies, where it participates in both tumorigenesis and cancer cell response to therapies. This is the case for Acute Promyelocytic Leukemias (APL) where SUMOylation, and subsequent destruction, of the PML-RARα fusion oncoprotein are triggered by arsenic trioxide, which is used as front-line therapy in combination with retinoic acid to cure APL patients. A similar arsenic-induced SUMO-dependent degradation was also documented for Tax, a human T-cell lymphotropic virus type I (HTLV1) viral protein implicated in Adult T-cell Leukemogenesis. SUMOylation also participates in Acute Myeloid Leukemia (AML) response to both chemo- and differentiation therapies, in particular through its ability to regulate gene expression. In Multiple Myeloma, many enzymes of the SUMO pathway are overexpressed and their high expression correlates with lower response to melphalan-based chemotherapies. B-cell lymphomas overexpressing the c-Myc oncogene also overexpress most components of the SUMO pathway and are highly sensitive to SUMOylation inhibition. Targeting the SUMO pathway with recently discovered pharmacological inhibitors, alone or in combination with current therapies, might therefore constitute a powerful strategy to improve the treatment of these cancers.

AP-1 Signaling by Fra-1 Directly Regulates HMGA1 Oncogene Transcription in Triple-Negative Breast Cancers.

The architectural chromatin protein HMGA1 and the transcription factor Fra-1 are both overexpressed in aggressive triple-negative breast cancers (TNBC), where they both favor epithelial-to-mesenchymal transition, invasion, and metastasis. We therefore explored the possibility that Fra-1 might be involved in enhanced transcription of the HMGA1 gene in TNBCs by exploiting cancer transcriptome datasets and resorting to functional studies combining RNA interference, mRNA and transcriptional run-on assays, chromatin immunoprecipitation, and chromosome conformation capture approaches in TNBC model cell lines. Our bioinformatic analysis indicated that Fra-1 and HMGA1 expressions positively correlate in primary samples of patients with TNBC. Our functional studies showed that Fra-1 regulates HMGA1 mRNA expression at the transcriptional level via binding to enhancer elements located in the last two introns of the gene. Although Fra-1 binding is required for p300/CBP recruitment at the enhancer domain, this recruitment did not appear essential for Fra-1-stimulated HMGA1 gene expression. Strikingly, Fra-1 binding is required for efficient recruitment of RNA Polymerase II at the HMGA1 promoter. This is permitted owing to chromatin interactions bringing about the intragenic Fra-1-binding enhancers and the gene promoter region. Fra-1 is, however, not instrumental for chromatin loop formation at the HMGA1 locus but rather exerts its transcriptional activity by exploiting chromatin interactions preexisting to its binding. IMPLICATIONS: We demonstrate that Fra-1 bound to an intragenic enhancer region is required for RNA Pol II recruitement at the HMGA1 promoter. Thereby, we provide novel insights into the mechanisms whereby Fra-1 exerts its prooncogenic transcriptional actions in the TNBC pathologic context.

Targeting Myeloperoxidase Disrupts Mitochondrial Redox Balance and Overcomes Cytarabine Resistance in Human Acute Myeloid Leukemia.

Chemotherapies alter cellular redox balance and reactive oxygen species (ROS) content. Recent studies have reported that chemoresistant cells have an increased oxidative state in hematologic malignancies. In this study, we demonstrated that chemoresistant acute myeloid leukemia (AML) cells had a lower level of mitochondrial and cytosolic ROS in response to cytarabine (AraC) and overexpressed myeloperoxidase (MPO), a heme protein that converts hydrogen peroxide to hypochlorous acid (HOCl), compared with sensitive AML cells. High MPO-expressing AML cells were less sensitive to AraC in vitro and in vivo. They also produced higher levels of HOCl and exhibited an increased rate of mitochondrial oxygen consumption when compared with low MPO-expressing AML cells. Targeting MPO expression or enzyme activity sensitized AML cells to AraC treatment by triggering oxidative damage and sustaining oxidative stress, particularly in high MPO-expressing AML cells. This sensitization stemmed from mitochondrial superoxide accumulation, which impaired oxidative phosphorylation and cellular energetic balance, driving apoptotic death and selective eradication of chemoresistant AML cells in vitro and in vivo. Altogether, this study uncovers a noncanonical function of MPO enzyme in maintaining redox balance and mitochondrial energetic metabolism, therefore affecting downstream pathways involved in AML chemoresistance. SIGNIFICANCE: These findings demonstrate the role of myeloperoxidase in the regulation of ROS levels and sensitivity of AML cells to cytarabine, an essential chemotherapeutic backbone in the therapy of AML.

The AP-1 transcriptional complex: Local switch or remote command?

The ubiquitous family of AP-1 dimeric transcription complexes is involved in virtually all cellular and physiological functions. It is paramount for cells to reprogram gene expression in response to cues of many sorts and is involved in many tumorigenic processes. How AP-1 controls gene transcription has largely remained elusive till recently. The advent of the "omics" technologies permitting genome-wide studies of transcription factors has however changed and improved our view of AP-1 mechanistical actions. If these studies confirm that AP-1 can sometimes act as a local transcriptional switch operating in the vicinity of transcription start sites (TSS), they strikingly indicate that AP-1 principally operates as a remote command binding to distal enhancers, placing chromatin architecture dynamics at the heart of its transcriptional actions. They also unveil novel constraints operating on AP-1, as well as novel mechanisms used to regulate gene expression via transcription-pioneering-, chromatin-remodeling- and chromatin accessibility maintenance effects.

Neutrophils are essential for induction of vaccine-like effects by antiviral monoclonal antibody immunotherapies.

Using a mouse retroviral model, we have shown that mAb-based immunotherapy can induce life-long endogenous protective immunity (vaccine-like effects). This observation has potentially important consequences for treating life-threatening human viral infections. Here, we investigated the role of neutrophils in this effect. Neutrophils are innate immunity effector cells with well-established microbe-killing activities that are rapidly mobilized upon infection. They are also emerging as orchestrators of innate and adaptive immunities. However, their immunomodulatory activity during antiviral mAb immunotherapies has never been studied. Our data reveal that neutrophils have an essential role in immunotherapy-induced immune protection of infected mice. Unexpectedly, neutrophils have a limited effect in controlling viral propagation upon passive immunotherapy administration, which is mostly mediated by NK cells. Instead, neutrophils operate as essential inducers of a potent host humoral antiviral response. Thus, neutrophils play an unexpected key role in protective immunity induction by antiviral mAbs. Our work opens approaches to improve antiviral immunotherapies, as it suggests that preserving neutrophil functions and counts might be required for achieving mAb-induced protective immunity.

Targeting the SUMO Pathway Primes All-trans Retinoic Acid-Induced Differentiation of Nonpromyelocytic Acute Myeloid Leukemias.

Differentiation therapies using all-trans retinoic acid (ATRA) are highly efficient at treating acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, their efficacy, if any, is limited in the case of non-APL AML. We report here that inhibition of SUMOylation, a posttranslational modification related to ubiquitination, restores the prodifferentiation and antiproliferative activities of retinoids in non-APL AML. Controlled inhibition of SUMOylation with the pharmacologic inhibitors 2-D08 or anacardic acid, or via overexpression of SENP deSUMOylases, enhanced the ATRA-induced expression of key genes involved in differentiation, proliferation, and apoptosis in non-APL AML cells. This activated ATRA-induced terminal myeloid differentiation and reduced cell proliferation and viability, including in AML cells resistant to chemotherapeutic drugs. Conversely, enhancement of SUMOylation via overexpression of the SUMO-conjugating enzyme Ubc9 dampened expression of ATRA-responsive genes and prevented differentiation. Thus, inhibition of the SUMO pathway is a promising strategy to sensitize patients with non-APL AML to retinoids and improve the treatment of this poor-prognosis cancer.Significance: SUMOylation silences key ATRA-responsive genes in nonpromyelocytic acute myeloid leukemias. Cancer Res; 78(10); 2601-13. ©2018 AACR.

Detection of Protein-Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway.

The detection of protein-protein interactions by imaging techniques often requires the overexpression of the proteins of interest tagged with fluorescent molecules, which can affect their biological properties and, subsequently, flaw experiment interpretations. The recent development of the proximity ligation assays (PLA) technology allows easy visualization of endogenous protein-protein interactions at the single molecule level. PLA relies on the use of combinations of antibodies coupled to complementary oligonucleotides that are amplified and revealed with a fluorescent probe, each spot representing a single protein-protein interaction. Another application of this technique is the detection of proteins posttranslational modifications to monitor their localization and dynamics in situ. Here, we describe the use of PLA to detect protein SUMOylation, a posttranslational modification related to ubiquitination, as well as interaction of SUMOylated substrates with other proteins, using both adherent and suspension cells.

Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria.

SUMO is a ubiquitin-like protein that is covalently conjugated to numerous cellular proteins to modify their function and fate. Although large progresses have been made in the identification of SUMOylated proteins, the molecular consequences of their SUMOylation are generally unknown. This is, most often, due to the low abundance of SUMOylated proteins in the cell, usually less than 1 % of a given protein being modified at steady state. To gain insights into the role of specific SUMOylation targets, SUMO conjugation can be reconstituted in vitro using purified proteins. However, for most substrates, the efficiency of in vitro SUMOylation is too low to obtain sufficient amounts of their SUMOylated forms for biochemical studies. Here, we describe a detailed protocol to purify large amounts of recombinant SUMOylated proteins using bacteria modified to express His-tagged SUMO as well as the SUMO-activating and -conjugating enzymes.

Converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play.

Monoclonal antibodies (mAbs), which currently constitute the main class of biotherapeutics, are now recognized as major medical tools that are increasingly being considered to fight severe viral infections. Indeed, the number of antiviral mAbs developed in recent years has grown exponentially. Although their direct effects on viral blunting have been studied in detail, their potential immunomodulatory actions have been overlooked until recently. The ability of antiviral mAbs to modulate antiviral immune responses in infected organisms has recently been revealed. More specifically, upon recognition of their cognate antigens, mAbs form immune complexes (ICs) that can be recognized by the Fc receptors expressed on different immune cells of infected individuals. This binding may be followed by the modulation of the host immune responses. Harnessing this immunomodulatory property may facilitate improvements in the therapeutic potential of antiviral mAbs. This review focuses on the role of ICs formed with different viral determinants and mAbs in the induction of antiviral immune responses in the context of both passive immunotherapies and vaccination strategies. Potential deleterious effects of ICs on the host immune response are also discussed.

c-Fos Protects Neurons Through a Noncanonical Mechanism Involving HDAC3 Interaction: Identification of a 21-Amino Acid Fragment with Neuroprotective Activity.

Proteins belonging to the AP-1 family of transcription factors are known to be involved in the regulation of neuronal viability. While strides have been made to elucidate the mechanisms of how individual members regulate cell death, much remains unknown. We find that the expression of one AP-1 member, c-Fos, is reduced in cerebellar granule neurons (CGNs) induced to die by low potassium (LK) treatment. Restoration and increase of this expression protect CGNs against LK-induced death, whereas knockdown induces death of otherwise healthy neurons. Furthermore, forced expression can protect cortical neurons against homocysteic acid (HCA)-induced toxicity. Taken together, this suggests that c-Fos is necessary for neuronal survival and that elevating c-Fos expression has a neuroprotective effect. Consistent with this idea is the finding that c-Fos expression is reduced selectively in the striatum in two separate mouse models of Huntington's disease and forced expression protects against neuronal death resulting from mutant huntingtin (mut-Htt) expression. Interestingly, neuroprotection by c-Fos does not require its DNA-binding, transcriptional, or heteromerization domains. However, this protective activity can be inhibited by pharmacological inhibition of c-Abl, CK-I, and MEK-ERK signaling. Additionally, expression of point mutant forms of this protein has identified that mutation of a tyrosine residue, Tyr345, can convert c-Fos from neuroprotective to neurotoxic. We show that c-Fos interacts with histone deacetylase-3 (HDAC3), a protein that contributes to mut-Htt neurotoxicity and whose overexpression is sufficient to promote neuronal death. When co-expressed, c-Fos can protect against HDAC3 neurotoxicity. Finally, our study identifies a 21-amino acid region at the C-terminus of c-Fos that is sufficient to protect neurons against death induced by LK, HCA treatment, or mut-Htt expression when expressed via a plasmid transfection or as a cell-permeable peptide. This cell-permeable peptide, designated as Fos-CTF, could have potential as a therapeutic agent for neurodegenerative diseases.

Antiviral Monoclonal Antibodies: Can They Be More Than Simple Neutralizing Agents?

Monoclonal antibodies (mAbs) are increasingly being considered as agents to fight severe viral diseases. So far, they have essentially been selected and used on the basis of their virus-neutralizing activity and/or cell-killing activity to blunt viral propagation via direct mechanisms. There is, however, accumulating evidence that they can also induce long-lasting protective antiviral immunity by recruiting the endogenous immune system of infected individuals during the period of immunotherapy. Exploiting this property may revolutionize antiviral mAb-based immunotherapies, with benefits for both patients and healthcare systems.