Open-label, phase 2 study of blinatumomab after frontline R-chemotherapy in adults with newly diagnosed, high-risk DLBCL.

This open-label, multicenter, single-arm, phase 2 study assessed the safety and efficacy of blinatumomab consolidation therapy in adult patients with newly diagnosed, high-risk diffuse large B-cell lymphoma (DLBCL; International Prognostic Index 3-5 and/or double-/triple-hit or double MYC/BCL-2 expressors) who achieved complete response (CR), partial response (PR), or stable disease (SD) following run-in with 6 cycles of R-chemotherapy (NCT03023878). Of the 47 patients enrolled, 28 received blinatumomab. Five patients (17.9%) experienced grade 4 treatment-emergent adverse events of interest (neutropenia, n = 4; infection, n = 1). Two deaths reported at the end of the study were unrelated to treatment with blinatumomab (disease progression, n = 1; infection, n = 1). 3/4 patients with PR and 4/4 patients with SD after R-chemotherapy achieved CR following blinatumomab. Consolidation with blinatumomab in patients with newly diagnosed, high-risk DLBCL who did not progress under R-chemotherapy was better tolerated than in previous studies where blinatumomab was used for treatment of patients with lymphoma.

TALE homeodomain proteins regulate site-specific terminal differentiation, LCE genes and epidermal barrier.

The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). We demonstrate here that a complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. We conclude that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers.

Whole-mount assays for gene induction and barrier formation in the developing epidermis.

The skin as a surface organ is uniquely accessible for whole embryo/foetal analyses of developmental changes, such as gene induction, protein expression, formation of epidermal-derived appendages such as hair follicles and formation of the protective barrier. Such analyses have emphasised the heterogeneous nature of skin development, perhaps not surprisingly because epidermal development is programmed by heterogeneous underlying mesenchyme. It is necessary to account for this heterogeneity by precisely matching body sites when correlating sequential events during development, for example, the activation of gene expression, or comparing wild-type with mutant/knockout animals. In this chapter protocols designed to assay whole-mount in situ hybridisation and whole-mount barrier formation are presented. Formation of the protective barrier is the endpoint of epidermal terminal differentiation and defects in this process are reflected in failure, acceleration, or delay in barrier formation. Hence, these latter assays are of particular value as a rapid initial assay for epidermal developmental defects in genetically modified mice.

AKT-dependent HspB1 (Hsp27) activity in epidermal differentiation.

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.

Late cornified envelope family in differentiating epithelia--response to calcium and ultraviolet irradiation.

The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum proteins. Within the LCE cluster, genes form "groups" based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two "skin" expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond "group-wise" to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.

Desmosomal cadherin misexpression alters beta-catenin stability and epidermal differentiation.

Desmosomal adhesion is important for the integrity and protective barrier function of the epidermis and is disregulated during carcinogenesis. Strong adhesion between keratinocytes is conferred by the desmosomal cadherins, desmocollin (Dsc) and desmoglein. These constitute two gene families, members of which are differentially expressed in epidermal strata. It has been suggested that this stratum-specific expression regulates keratinocyte differentiation. We tested this hypothesis by misdirecting the expression of the basally abundant desmosomal cadherins Dsc3a and Dsc3b to suprabasal differentiating keratinocytes in transgenic mice. No phenotype was apparent until adulthood, when mice developed variable ventral alopecia and had altered keratinocyte differentiation within affected areas. The follicular changes were reminiscent of changes in transgenic mice with an altered beta-catenin stability. Stabilized beta-catenin and increased beta-catenin transcriptional activity were demonstrated in transgenic mice prior to the phenotypic change and in transgenic keratinocytes as a consequence of transgene expression. Hence, a link between desmosomal cadherins and beta-catenin stability and signaling was demonstrated, and it was shown that desmocollin cadherin expression can affect keratinocyte differentiation. Furthermore, the first function for a "b-type" desmocollin cadherin was demonstrated.

Multipotent cell lineages in early mouse development depend on SOX2 function.

Each cell lineage specified in the preimplantation mammalian embryo depends on intrinsic factors for its development, but there is also mutual interdependence between them. OCT4 is required for the ICM/epiblast lineage, and at transient high levels for extraembryonic endoderm, but also indirectly through its role in regulating Fgf4 expression, for the establishment and proliferation of extraembryonic ectoderm from polar trophectoderm. The transcription factor SOX2 has also been implicated in the regulation of Fgf4 expression. We have used gene targeting to inactivate Sox2, examining the phenotypic consequences in mutant embryos and in chimeras in which the epiblast is rescued with wild-type ES cells. We find a cell-autonomous requirement for the gene in both epiblast and extraembryonic ectoderm, the multipotent precursors of all embryonic and trophoblast cell types, respectively. However, an earlier role within the ICM may be masked by the persistence of maternal protein, whereas the lack of SOX2 only becomes critical in the chorion after 7.5 days postcoitum. Our data suggest that maternal components could be involved in establishing early cell fate decisions and that a combinatorial code, requiring SOX2 and OCT4, specifies the first three lineages present at implantation.