A chemogenomic approach to identify personalized therapy for patients with relapse or refractory acute myeloid leukemia: results of a prospective feasibility study.

Targeted next-generation sequencing (tNGS) and ex vivo drug sensitivity/resistance profiling (DSRP) have laid foundations defining the functional genomic landscape of acute myeloid leukemia (AML) and premises of personalized medicine to guide treatment options for patients with aggressive and/or chemorefractory hematological malignancies. Here, we have assessed the feasibility of a tailored treatment strategy (TTS) guided by systematic parallel ex vivo DSRP and tNGS for patients with relapsed/refractory AML (number NCT02619071). A TTS issued by an institutional personalized committee could be achieved for 47/55 included patients (85%), 5 based on tNGS only, 6 on DSRP only, while 36 could be proposed on the basis of both, yielding more options and a better rationale. The TSS was available in <21 days for 28 patients (58.3%). On average, 3 to 4 potentially active drugs were selected per patient with only five patient samples being resistant to the entire drug panel. Seventeen patients received a TTS-guided treatment, resulting in four complete remissions, one partial remission, and five decreased peripheral blast counts. Our results show that chemogenomic combining tNGS with DSRP to determine a TTS is a promising approach to propose patient-specific treatment options within 21 days.

Nectin-4: a new prognostic biomarker for efficient therapeutic targeting of primary and metastatic triple-negative breast cancer.

Background: Triple-negative breast cancers (TNBCs) are associated with a poor prognosis. In contrast to other molecular subtypes, they have no identified specific target and chemotherapy remains the only available systemic treatment. The adhesion molecule nectin-4 represents a new potential therapeutic target in different cancer models. Here, we have tested the prognostic value of nectin-4 expression and assessed the therapeutic efficiency of an anti-nectin 4 antibody drug conjugate (ADC) on localised and metastatic TNBC in vitro and in vivo. Materials and methods: We analysed nectin-4/PVRL4 mRNA expression in 5673 invasive breast cancers and searched for correlations with clinicopathological features including metastasis-free survival (MFS). Immunohistochemistry was carried out in 61 TNBCs and in samples of primary TNBC Patient-Derived Xenografts (PDXs). An anti-nectin-4 antibody eligible for ADC was produced and tested in vitro and in vivo in localised and metastatic TNBC PDXs. Results: High nectin-4/PVRL4 mRNA expression was associated with poor-prognosis features including the TN and basal subtypes. High PVRL4 mRNA expression showed independent negative prognostic value for MFS in multivariate analysis in TNBCs. Nectin-4 protein expression was not detected in adult healthy tissues including mammary tissue. Membranous protein expression was found in 62% of TNBCs, with strong correlation with mRNA expression. We developed an ADC (N41mab-vcMMAE) comprising a human anti-nectin-4 monoclonal antibody conjugated to monomethyl auristatin-E (MMAE). In vitro, this ADC bound to nectin-4 with high affinity and specificity and induced its internalisation as well as dose-dependent cytotoxicity on nectin-4-expressing breast cancer cell lines. In vivo, this ADC induced rapid, complete and durable responses on nectin-4-positive xenograft TNBC samples including primary tumours, metastatic lesions, and local relapses; efficiency was dependent on both the dose and the nectin-4 tumour expression level. Conclusion: Nectin-4 is both a new promising prognostic biomarker and specific therapeutic target for ADC in the very limited armamentarium against TNBC.

A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia.

Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2Rγc(null) mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies.

Anti-leukemia activity of chaetocin via death receptor-dependent apoptosis and dual modulation of the histone methyl-transferase SUV39H1.

Epigenetic deregulation is involved in acute myeloid leukemia (AML) pathogenesis and epigenetic targeting drugs are in clinical trial. Since the first results with histone-deacetylase inhibitors in AML are controversial, novel single and combined treatments need to be explored. It is tempting to combine chromatin-targeting drugs. SUV39H1, the main methyl-transferase for lysine 9 tri-methylation on histone H3, interacts with oncogenes involved in AML and acts as a transcriptional repressor for hematopoietic differentiation and immortalization. We report here that pharmacological inhibition of SUV39H1 by chaetocin induces apoptosis in leukemia cell lines in vitro and primary AML cells ex vivo, and that it interferes with leukemia growth in vivo. Chaetocin treatment upregulates reactive oxygen species (ROS) production as well as the transcription of death-receptor-related genes, in a ROS-dependent manner, leading to death receptor-dependent apoptosis. In addition to its direct inhibition by chaetocin, SUV39H1 is indirectly modulated by chaetocin-induced ROS. Accordingly, chaetocin potentiates other anti-AML drugs, in a ROS-dependent manner. The decryption of a dual mechanism of action against AML involving both direct and indirect SUV39H1 modulation represents an innovative read-out for the anticancer activity of chaetocin and for its synergy with other anti-AML drugs, suggesting new therapeutic combination strategies in AML.

Alternative splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn.

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.

Interaction with simian Hck tyrosine kinase reveals convergent evolution of the Nef protein from simian and human immunodeficiency viruses despite differential molecular surface usage.

Simian and human immunodeficiency virus type 1 (SIV and HIV-1) Nef proteins are thought to use different molecular surfaces to mediate the protein-protein interactions required for their otherwise similar functions. This genetically separable function suggests convergent evolution of primate lentiviruses and/or structural differences between human and nonhuman primate cellular target proteins. However, such comparative molecular analyses have not been undertaken so far using the respective natural host-derived cellular targets. We cloned simian Src family kinase Hck and analyzed structurally and biochemically its interaction with SIV Nef.

The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral vif protein.

The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

Role of Tec kinase in nuclear factor of activated T cells signaling.

The Tec protein kinase family includes Btk, Itk, Tec, Rlk and Bmx, which are critically involved in signals mediated by various cytokines and antigen receptors. Btk mutations cause severe immunodeficiencies, with defective B cell function. In T cells, Tec regulates cytokine production. However, the downstream targets of these Tec kinases are poorly defined. Here we report that overexpression of Tec in T cells can regulate gene transcription through the nuclear factor of activated T cells (NF-AT). Using different reporter gene constructs, we establish that Tec in transfected T cells dramatically induced NF-AT-dependent gene transcription, which was prevented by a dominant-negative mutant of NF-AT or by the immunosuppressive drug cyclosporin A. Tec appears to regulate NF-AT nuclear import. In addition, Tec influences cytoplasmic free calcium increase. Taken together, our results identify NF-AT as a major downstream target of Tec kinases that is critically involved in transcriptional gene regulation. These observations highlight signaling pathways regulated by Tec kinases and provide new pharmacological targets to regulate immune functions.

Parameters involved in the recognition of fresh human leukemic blasts by tumor-specific cytolytic T cell clones: a model study.

Although clinical experience and in vitro data provide evidence of an anti-leukemic activity of T cells, there are few examples of recognition of leukemic cells by tumor-specific T cells in vitro. Tumor antigens encoded by the MAGE genes are useful tools to study this recognition. We tested the sensitivity to recognition and lysis by anti-MAGE CTL clones of MAGE-A1 positive cell lines HL60 and K562, after transfection with an HLA-A1 construct, and of fresh leukemic blasts from 10 HLA-A2 patients, after incubation with a peptide encoded by gene MAGE-A3. The presentation of MAGE antigens by leukemic cell lines and fresh leukemic blasts induced TNF secretion and cytotoxicity by MAGE-specific CD8(+) CTL clones. The amount of peptide presented by the leukemic blasts, more than the level of expression of HLA class I, adhesion or costimulatory molecules, was the major limiting factor for recognition. These data indicate that leukemic cells may be targeted by T cells showing specificity for a leukemia antigen.

The primate lentivirus-encoded Nef protein can regulate several steps of the viral replication cycle.

The primate lentiviruses encode a protein, Nef, which is required for efficient viral replication in their host. Several biological activities of nef identified in vitro may contribute to this requirement in vivo, including receptor modulation and interference with cellular signaling pathways. We show that HIV- and SIV-encoded Nef can enhance virus production within a single viral replication cycle, not only by increasing viral infectivity, as previously reported, but also by acting through the efficiency of viral transcription and of viral release.

Simian immunodeficiency virus and human immunodeficiency virus type 1 nef proteins show distinct patterns and mechanisms of Src kinase activation.

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.

Human immunodeficiency virus type 1 Nef protein sensitizes CD4(+) T lymphoid cells to apoptosis via functional upregulation of the CD95/CD95 ligand pathway.

Many viruses have evolved genes encoding proteins that regulate cell death by apoptosis. The human immunodeficiency virus type 1 (HIV-1) Nef protein alters T-cell development and signaling and is required for optimal viral replication and pathogenicity in vivo. To analyze the interference of Nef with cell survival, we used both regulated and constitutively expressed nef alleles in stably transfected T-cell lines. Nef-expressing cells were sensitized to cell death by apoptosis, which was specifically exacerbated by an anti-CD95 IgM monoclonal antibody (MoAb). Flow cytometric analysis showed that the surface expression of both CD95 and CD95 ligand (CD95L) was upregulated by endogenous Nef expression. Nef-mediated apoptosis was almost completely suppressed by the addition in culture of an anti-CD95 Fab' IgG MoAb, which specifically blocks CD95/CD95L interactions. Lastly, mutation of a proline motif in the core region of the nef gene, which disrupts its ability to interact with cellular kinases and reduces HIV-1 replication in vitro, completely abrogated the Nef-mediated induction of apoptosis as well as its ability to upregulate surface CD95 and CD95L. These findings may provide molecular insight into the role of endogenous Nef in the T-cell depletion observed in vivo, particularly HIV-specific cytotoxic CD8(+) T cells.

The human immunodeficiency virus type 1 Nef protein binds the Src-related tyrosine kinase Lck SH2 domain through a novel phosphotyrosine independent mechanism.

Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein.

Distinct regulation of T-cell death by CD28 depending on both its aggregation and T-cell receptor triggering: a role for Fas-FasL.

CD28 is a major coreceptor that regulates cell proliferation, anergy, and viability of T cells. The negative selection by T-cell receptor (TCR)-induced cell death of immature thymocytes as well as of activated human antigen-specific T-cell clone, requires a costimulatory signal that can be provided by CD28. Conversely, CD28-mediated signals increase expression of Bcl-XL, a survival gene, and promote survival of naive T cells cultured in the absence of antigen or costimulation. Because CD28 appears to both protect from, or induce T-cell death, one important question is to define the activation and cellular parameters that dictate the differential role of CD28 in T-cell apoptosis. Here, we compared different CD28 ligands for their ability to regulate TCR-induced cell death of a murine T-cell hybridoma. In these cells, TCR triggering induced expression of Fas and FasL, and cell death was prevented by anti-Fas blocking monoclonal antibody (MoAb). When provided as a costimulus, both CD28 MoAb and the B7.1 and B7.2 counter receptors downregulated, yet did not completely abolish T-cell receptor-induced apoptosis. This CD28 cosignal resulted in both upregulation of Bcl-XL and prevention of FasL expression. In marked contrast, when given as a single signal, CD28 MoAb or B7.1 and B7.2 induced FasL expression and resulted in T-cell death by apoptosis, which was dependent on the level of CD28 ligation. Furthermore, triggering of CD28 upregulated FasL and induced a marked T-cell death of previously activated normal peripheral T cells. Our results identify Fas and FasL as crucial targets of CD28 in T-cell death regulation and show that within the same cell population, depending on its engagement as a single signal or as a costimulus together with the TCR, CD28 can either induce a dose-dependent death signal or protect from cell death, respectively. These data provide important insights into the role of CD28 in T-cell homeostasis and its possible implication in neoplastic disorders.