Autologous T cell therapy for MAGE-A4+ solid cancers in HLA-A*02+ patients: a phase 1 trial.

Affinity-optimized T cell receptors can enhance the potency of adoptive T cell therapy. Afamitresgene autoleucel (afami-cel) is a human leukocyte antigen-restricted autologous T cell therapy targeting melanoma-associated antigen A4 (MAGE-A4), a cancer/testis antigen expressed at varying levels in multiple solid tumors. We conducted a multicenter, dose-escalation, phase 1 trial in patients with relapsed/refractory metastatic solid tumors expressing MAGE-A4, including synovial sarcoma (SS), ovarian cancer and head and neck cancer ( NCT03132922 ). The primary endpoint was safety, and the secondary efficacy endpoints included overall response rate (ORR) and duration of response. All patients (N = 38, nine tumor types) experienced Grade ≥3 hematologic toxicities; 55% of patients (90% Grade ≤2) experienced cytokine release syndrome. ORR (all partial response) was 24% (9/38), 7/16 (44%) for SS and 2/22 (9%) for all other cancers. Median duration of response was 25.6 weeks (95% confidence interval (CI): 12.286, not reached) and 28.1 weeks (95% CI: 12.286, not reached) overall and for SS, respectively. Exploratory analyses showed that afami-cel infiltrates tumors, has an interferon-γ-driven mechanism of action and triggers adaptive immune responses. In addition, afami-cel has an acceptable benefit-risk profile, with early and durable responses, especially in patients with metastatic SS. Although the small trial size limits conclusions that can be drawn, the results warrant further testing in larger studies.

Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcoma.

BACKGROUND: Gene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells. METHODS: Four cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay. RESULTS: Responses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients. CONCLUSIONS: Our studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01343043 , Registered 27 April 2011.

Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 c259T Cells in Synovial Sarcoma.

We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR.See related commentary by Keung and Tawbi, p. 914This article is highlighted in the In This Issue feature, p. 899.

Distinct gene expression program dynamics during erythropoiesis from human induced pluripotent stem cells compared with adult and cord blood progenitors.

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) are a potentially invaluable resource for regenerative medicine, including the in vitro manufacture of blood products. HiPSC-derived red blood cells are an attractive therapeutic option in hematology, yet exhibit unexplained proliferation and enucleation defects that presently preclude such applications. We hypothesised that substantial differential regulation of gene expression during erythroid development accounts for these important differences between hiPSC-derived cells and those from adult or cord-blood progenitors. We thus cultured erythroblasts from each source for transcriptomic analysis to investigate differential gene expression underlying these functional defects. RESULTS: Our high resolution transcriptional view of definitive erythropoiesis captures the regulation of genes relevant to cell-cycle control and confers statistical power to deploy novel bioinformatics methods. Whilst the dynamics of erythroid program elaboration from adult and cord blood progenitors were very similar, the emerging erythroid transcriptome in hiPSCs revealed radically different program elaboration compared to adult and cord blood cells. We explored the function of differentially expressed genes in hiPSC-specific clusters defined by our novel tunable clustering algorithms (SMART and Bi-CoPaM). HiPSCs show reduced expression of c-KIT and key erythroid transcription factors SOX6, MYB and BCL11A, strong HBZ-induction, and aberrant expression of genes involved in protein degradation, lysosomal clearance and cell-cycle regulation. CONCLUSIONS: Together, these data suggest that hiPSC-derived cells may be specified to a primitive erythroid fate, and implies that definitive specification may more accurately reflect adult development. We have therefore identified, for the first time, distinct gene expression dynamics during erythroblast differentiation from hiPSCs which may cause reduced proliferation and enucleation of hiPSC-derived erythroid cells. The data suggest several mechanistic defects which may partially explain the observed aberrant erythroid differentiation from hiPSCs.

A network including TGFß/Smad4, Gata2, and p57 regulates proliferation of mouse hematopoietic progenitor cells.

Transforming growth factor ß (TGFß) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell function, as an early and direct target gene for TGFß-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFß response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFß signaling effector molecule p57 was found to be upregulated as a secondary response to TGFß. We observed Gata2 binding upstream of the p57 genomic locus, and importantly, loss of Gata2 abolished TGFß-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in hematopoietic stem cell self-renewal and reveal a functionally relevant network, regulating proliferation of primitive hematopoietic cells.

Distinct mechanisms of inadequate erythropoiesis induced by tumor necrosis factor alpha or malarial pigment.

The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10 ng/ml) or to hemozoin (12.5 µg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.

Dynamic analysis of gene expression and genome-wide transcription factor binding during lineage specification of multipotent progenitors.

We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.

Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments.

Gene expression in multiple individual cells from a tissue or culture sample varies according to cell-cycle, genetic, epigenetic and stochastic differences between the cells. However, single-cell differences have been largely neglected in the analysis of the functional consequences of genetic variation. Here we measure the expression of 92 genes affected by Wnt signaling in 1,440 single cells from 15 individuals to associate single-nucleotide polymorphisms (SNPs) with gene-expression phenotypes, while accounting for stochastic and cell-cycle differences between cells. We provide evidence that many heritable variations in gene function--such as burst size, burst frequency, cell cycle-specific expression and expression correlation/noise between cells--are masked when expression is averaged over many cells. Our results demonstrate how single-cell analyses provide insights into the mechanistic and network effects of genetic variability, with improved statistical power to model these effects on gene expression.

Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells.

The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR.

Inferring rules of lineage commitment in haematopoiesis.

How the molecular programs of differentiated cells develop as cells transit from multipotency through lineage commitment remains unexplored. This reflects the inability to access cells undergoing commitment or located in the immediate vicinity of commitment boundaries. It remains unclear whether commitment constitutes a gradual process, or else represents a discrete transition. Analyses of in vitro self-renewing multipotent systems have revealed cellular heterogeneity with individual cells transiently exhibiting distinct biases for lineage commitment. Such systems can be used to molecularly interrogate early stages of lineage affiliation and infer rules of lineage commitment. In haematopoiesis, population-based studies have indicated that lineage choice is governed by global transcriptional noise, with self-renewing multipotent cells reversibly activating transcriptome-wide lineage-affiliated programs. We examine this hypothesis through functional and molecular analysis of individual blood cells captured from self-renewal cultures, during cytokine-driven differentiation and from primary stem and progenitor bone marrow compartments. We show dissociation between self-renewal potential and transcriptome-wide activation of lineage programs, and instead suggest that multipotent cells experience independent activation of individual regulators resulting in a low probability of transition to the committed state.

GATA-2 mediated regulation of normal hematopoietic stem/progenitor cell function, myelodysplasia and myeloid leukemia.

Unremitting blood cell production throughout the lifetime of an organism is reliant on hematopoietic stem cells (HSCs). A rare and relatively quiescent cell type, HSCs are, on entry into cell cycle fated to self-renew, undergo apoptosis or differentiate to progenitors (HPCs) that eventually yield specific classes of blood cells. Disruption of these HSC fate decisions is considered to be fundamental to the development of leukemia. Much effort has therefore been placed on understanding the molecular pathways that regulate HSC fate decisions and how these processes are undermined in leukemia. Transcription factors have emerged as critical regulators in this respect. Here we review the participation of zinc finger transcription factor GATA-2 in regulating normal hematopoietic stem and progenitor cell functionality, myelodysplasia and myeloid leukemia.

High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle.

Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G(0) residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34(+)CD38(-)Hoechst(lo)Pyronin Y(lo)) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2-conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21(CIP1) and p27(KIP1) do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2(hi)) failed to contribute to hematopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2(lo) cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

GATA-2 regulates granulocyte-macrophage progenitor cell function.

The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein, we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2(+/-)) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin(-)CD117(+)Sca-1(-)CD34(+)CD16/32(high) granulocyte-macrophage progenitor (GMP) compartment of GATA-2(+/-) marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. Although apoptosis and cell-cycle distribution remained unperturbed in GATA-2(+/-) GMP, quiescent cells from GATA-2(+/-) GMP exhibited altered functionality. Gene expression analysis showed attenuated expression of HES-1 mRNA in GATA-2-deficient GMP. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3, and enforced expression of HES-1 expression in GATA-2(+/-) GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as a novel, pivotal determinant of GMP cell fate.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1.

Specific deletion of Notch1 and RBPjkappa in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

Novel compounds with antiproliferative activity against imatinib-resistant cell lines.

Chronic myelogenous leukemia is caused by the Bcr-Abl hybrid gene that encodes the p210Bcr-Abl chimeric oncoprotein. Although it reduces the total body burden of leukemia cells, the use of imatinib mesylate as a single agent may be accompanied by the evolution of resistance due mainly to the acquisition of point mutations. Imatinib has been combined with drugs that inhibit both the active and the inactive states of the p210Bcr-Abl kinase. These combinations have reduced but not completely eliminated the rate at which point mutations are acquired in the p210Bcr-Abl kinase. Thus, it is important to identify additional new inhibitors of the p210Bcr-Abl kinase. One possible method to prevent evolution of resistance is to simultaneously use multiple kinase inhibitors each with a different mechanism of action. To identify such a new class of inhibitors that could suppress the growth of chronic myelogenous leukemia cells and prevent the evolution of cells that are resistant to imatinib, we screened two low-complexity libraries of compounds based on planar and linear scaffolds. These libraries were screened using a cell-based assay for molecules that suppress p210Bcr-Abl-dependent cell growth. The application of this method resulted in the isolation of two new classes of drugs, both of which inhibited imatinib-resistant cells in the low micromolar range. Some of these drugs were potent inhibitors not only of Abl tyrosine kinase but also of the Src, Lyn, and Fyn tyrosine kinases.

ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

Modulation of the p38 MAPK (mitogen-activated protein kinase) pathway through Bcr/Abl: implications in the cellular response to Ara-C.

The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.