Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness.

The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

Genetically engineered T cells expressing a HER2-specific chimeric receptor mediate antigen-specific tumor regression.

In this report, we developed a chimeric receptor (N29gamma chR) involving the single chain Fv (scFv) derived from N29 monoclonal antibody (mAb) specific for p185HER2 and characterized the therapeutic efficacy of primary T cells engineered to express N29gamma chR in two histologically distinct murine tumor models. Murine breast (MT901) and fibrosarcoma (MCA207) cancer cell lines were engineered to express human HER2 as targets. Administration of N29gamma chR-expressing T cells eliminated 3-day pulmonary micrometastases of MT901/HER2 and MCA207/HER2 but not parental tumor cells. A 5 to 8-fold increased dose of N29gamma T cells was required to mediate regression of advanced 8-day macrometastases. Exogenous administration of interleukin-2 (IL-2) after N29gamma T-cell transfer was dispensable for treatment of 3-day micrometastases, but was required for mediating regression of well-established 8-day macrometastases. Moreover, fractionated CD8 T cells expressing N29gamma chR suppressed HER2-positive tumor cell growth after adoptive transfer independent of CD4(+) cells. These data indicate that genetically modified T cells expressing a HER2-targeting chimeric receptor can mediate antigen-specific regression of preestablished metastatic cancers in a cell dose-dependent fashion. Systemic administration of IL-2 augments the therapeutic efficacy of these genetically engineered T cells in advanced diseases. These results are relevant to the implication of genetically redirected T cells in clinical cancer immunotherapy.

Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo.

BACKGROUND: Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2. METHODS: We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively. RESULTS: In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P<0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer. DISCUSSION: We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.