FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

Rationally designed chimeric PI3K-BET bromodomain inhibitors elicit curative responses in MYC-driven lymphoma.

Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited antilymphoma activity as single agents in the context of cellular Myelocytomatosis (cMYC) oncogene-dysregulation. However, combined PI3K and BET inhibition imparts synergistic anticancer activity with the potential for more sustained disease responses due to the mutual antagonism of compensatory epigenetic and signaling networks. Here, we describe the mechanistic and therapeutic validation of rationally designed dual PI3K/BET bromodomain inhibitors, built by linkage of established PI3K and BET inhibitor pharmacophores. The lead candidate demonstrates high selectivity, nanomolar range cellular potency, and compelling in vivo efficacy, including curative responses in the aggressive Eµ-Myc lymphoma model. These studies further support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential step-change in approach to orthogonal MYC antagonism using optimized chimeric small-molecule technology.

The curious case of IDH mutant acute myeloid leukaemia: biochemistry and therapeutic approaches.

Of the many genetic alterations that occur in cancer, relatively few have proven to be suitable for the development of targeted therapies. Mutations in isocitrate dehydrogenase (IDH) 1 and -2 increase the capacity of cancer cells to produce a normally scarce metabolite, D-2-hydroxyglutarate (2-HG), by several orders of magnitude. The discovery of the unusual biochemistry of IDH mutations spurred a flurry of activity that revealed 2-HG as an 'oncometabolite' with pleiotropic effects in malignant cells and consequences for anti-tumour immunity. Over the next decade, we learned that 2-HG dysregulates a wide array of molecular pathways, among them a large family of dioxygenases that utilise the closely related metabolite α-ketoglutarate (α-KG) as an essential co-substrate. 2-HG not only contributes to malignant transformation, but some cancer cells become addicted to it and sensitive to inhibitors that block its synthesis. Moreover, high 2-HG levels and loss of wild-type IDH1 or IDH2 activity gives rise to synthetic lethal vulnerabilities. Herein, we review the biology of IDH mutations with a particular focus on acute myeloid leukaemia (AML), an aggressive disease where selective targeting of IDH-mutant cells is showing significant promise.

Inhibition of mutant IDH1 promotes cycling of acute myeloid leukemia stem cells.

Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.

Inhibition of pyrimidine biosynthesis targets protein translation in acute myeloid leukemia.

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

Integrated clinical and genomic evaluation of guadecitabine (SGI-110) in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a rare, heterogenous malignancy with dismal outcomes at relapse. Hypomethylating agents (HMA) have an emerging role in PTCL, supported by shared mutations with myelodysplasia (MDS). Response rates to azacitidine in PTCL of follicular helper cell origin are promising. Guadecitabine is a decitabine analogue with efficacy in MDS. In this phase II, single-arm trial, PTCL patients received guadecitabine on days 1-5 of 28-day cycles. Primary end points were overall response rate (ORR) and safety. Translational sub-studies included cell free plasma DNA sequencing and functional genomic screening using an epigenetically-targeted CRISPR/Cas9 library to identify response predictors. Among 20 predominantly relapsed/refractory patients, the ORR was 40% (10% complete responses). Most frequent grade 3-4 adverse events were neutropenia and thrombocytopenia. At 10 months median follow-up, median progression free survival (PFS) and overall survival (OS) were 2.9 and 10.4 months respectively. RHOAG17V mutations associated with improved PFS (median 5.47 vs. 1.35 months; Wilcoxon p = 0.02, Log-Rank p = 0.06). 4/7 patients with TP53 variants responded. Deletion of the histone methyltransferase SETD2 sensitised to HMA but TET2 deletion did not. Guadecitabine conveyed an acceptable ORR and toxicity profile; decitabine analogues may provide a backbone for future combinatorial regimens co-targeting histone methyltransferases.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Clonal multi-omics reveals Bcor as a negative regulator of emergency dendritic cell development.

Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.

Non-genetic heterogeneity, altered cell fate and differentiation therapy.

Altered capacity for self-renewal and differentiation is a hallmark of cancer, and many tumors are composed of cells with a developmentally immature phenotype. Among the malignancies where processes that govern cell fate decisions have been studied most extensively is acute myeloid leukemia (AML), a disease characterized by the presence of large numbers of "blasts" that resemble myeloid progenitors. Classically, the defining properties of AML cells were said to be aberrant self-renewal and a block of differentiation, and the term "differentiation therapy" was coined to describe drugs that promote the maturation of leukemic blasts. Notionally however, the simplistic view that such agents "unblock" differentiation is at odds with the cancer stem cell (CSC) hypothesis that posits that tumors are hierarchically organized and that CSCs, which underpin cancer growth, retain the capacity to progress to a developmentally more mature state. Herein, we will review recent developments that are providing unprecedented insights into non-genetic heterogeneity both at steady state and in response to treatment, and propose a new conceptual framework for therapies that aim to alter cell fate decisions in cancer.

Pharmacological and genetic strategies for targeting adenosine to enhance adoptive T cell therapy of cancer.

Adoptive cellular therapy involves the ex vivo expansion of immune cells, conventionally T cells, before reinfusion back to the patient. Variations in adoptive cellular therapy include transduction of a patient's T cells with either a transgenic T cell receptor or chimeric antigen receptor (CAR) to recognize a defined tumor antigen. Given that adenosine is a major axis of immunosuppression of T cells, particularly in hypoxic tumor microenvironments, therapeutics targeting this pathway are currently being assessed for their potential to enhance adoptive T cell therapies. The use of gene-editing technology, commonly used in tandem with CAR and transgenic T cell receptor (TCR) based adoptive cellular therapy, offers further opportunities to specifically modulate responses to adenosine. This review will discuss recent advances in targeting the adenosine pathway for enhancing the effectiveness of adoptive cellular therapy in the treatment of solid cancers.

IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.

Bcor loss perturbs myeloid differentiation and promotes leukaemogenesis.

The BCL6 Corepressor (BCOR) is a component of a variant Polycomb repressive complex 1 (PRC1) that is essential for normal development. Recurrent mutations in the BCOR gene have been identified in acute myeloid leukaemia and myelodysplastic syndrome among other cancers; however, its function remains poorly understood. Here we examine the role of BCOR in haematopoiesis in vivo using a conditional mouse model that mimics the mutations observed in haematological malignancies. Inactivation of Bcor in haematopoietic stem cells (HSCs) results in expansion of myeloid progenitors and co-operates with oncogenic KrasG12D in the initiation of an aggressive and fully transplantable acute leukaemia. Gene expression analysis and chromatin immunoprecipitation sequencing reveals differential regulation of a subset of PRC1-target genes including HSC-associated transcription factors such as Hoxa7/9. This study provides mechanistic understanding of how BCOR regulates cell fate decisions and how loss of function contributes to the development of leukaemia.

Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells.

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells.

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) - Isoleucine (I) - Lysine (K) - Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite's ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.

Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance.

Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2(R140Q) in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2(R140Q) can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.

Bioinformatic prediction of the exportome of Babesia bovis and identification of novel proteins in parasite-infected red blood cells.

Babesia bovis is a pathogen of considerable economic significance to the livestock industry worldwide but the precise mechanisms by which this parasite causes disease in susceptible cattle remain poorly understood. It is clear, however, that alterations to the structure and function of red blood cells in which the parasites reside and replicate play an important role in pathogenesis and that these are secondary to the export of numerous, currently unknown and uncharacterised parasite-encoded proteins. Using a rational bioinformatic approach, we have identified a set of 362 proteins (117 of which are hypothetical) that we predict encompasses the B. bovis exportome. These exported proteins are likely to be trafficked to various cellular locations, with a subset destined for the red blood cell cytosol or the red blood cell cytoskeleton. These proteins are likely to play important roles in mediating the pathogenesis of babesiosis. We have selected three novel proteins and confirmed their predicted export and localisation within the host red blood cell by immunofluorescence using specific antibodies raised against these proteins. Complete characterisation of these novel exported parasite proteins will help elucidate their function within the host red blood cell and assist in identification of new therapeutic targets for babesiosis.