GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia

Abstract

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18–24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

γ‐Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and notch down‐regulation

“…In precursor-B ALL cells, GSI I induces apoptosis by inhibiting Notch signaling and through Notchindependent mechanisms including, besides proteasome inhibition, the increase of reactive oxygen species production and the disruption of the AKT pro-survival pathway. 19 The ability of GSI I to target Notch, AKT and other pro-survival pathways, has also been shown in cutaneous T-cell lymphoma. 50 All these findings have led to an in-depth understanding of the mechanisms of action of GSI I in tumor cells which is essential to define the potential clinical use of GSI I.…”

“…11 However, in these malignancies, minimal clinical responses have been observed, and other studies have also suggested that Notch inhibition is therapeutically effective only when combined with inhibition of other relevant pathogenic pathways. [12][13][14] It has been recently shown that GSI I (Z-Leu-Leu-Nle-CHO), a tripeptide aldehyde inhibiting chymotryptic and aspartyl protease activity, chemically similar to the proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO), 15 induces apoptosis in breast 16,17 and glioblastoma tumor cells, 18 and in precursor-B acute lymphoblastic leukemia (ALL) cells 19 by inhibiting both c-secretase and proteasome activity or only proteasome activity. 17 The ability of GSI I to target both Notch and proteasome can render it more effective in inducing antitumor activity in those tumor cells where dysregulated survival and/ or growth are associated with alterations in both Notch signaling and proteasome activity.…”

“…9 Here, to define how this event contributes to GSI I-induced CLL cell apoptosis, we first examined the time-course effect of GSI I on Notch1 and Notch2 processing and Hes1 protein expression, a target of Notch2 in CLL cells. 9 We performed these experiments in CLL cells from 12 samples (2)(3)(4)(5)(6)(7)(17)(18)(19)(20)(21)(22), including patients with different characteristics. In all samples, GSI I inhibited the generation of Notch1 and Notch2 intracellular domains (IC), as well as the expression of the precursors (P) and the transmembrane/cytoplasmic portion (TM), only after 24 hr, whereas up to 8 hr, the levels of the three forms of both receptors appeared slightly increased or unchanged compared with controls (Fig.…”

Development of blood-brain barrier under the modulation of HIF activity in astroglial and neuronal cells in vitro

“…При предварительной инкубации микс-культуры нейронов и астроцитов с GSI-I в концентрации 100 нМ, что соответствует эффективной концентрации, не вызывающей значительного снижения жизнеспособности клеток [20], к третьим суткам мы обнаружили статистически значимое увеличение экспрессии TS1, TS2 и, в значительно большей степени, чем при действии ингибитора HIF-1 -MMP-9, на фоне уменьшения экспрессии VEGF (рис. 1Б).…”

“…1Б). Однако, с учётом того, что GSI-I способен подавлять убиквитин-протеасомный путь деградации белков [20], стимулирующий эффект этого модулятора на стабилизацию HIF-1 в клетках, находящихся в условиях нормоксии, не может быть исключён. С учётом однонаправленности изменений в экспрессии тромбоспондинов и матриксных металлопротеиназ при применении FM19G11 и GSI-I, логично предположить, что в клетках нейрональной и астроглиальной природы в модели формирующегося ГЭБ в условиях нормоксии FM19G11 может оказывать стимулирующее действие на экспрессию HIF-1 (по аналогии с данными [15]), и к такому же эффекту приводит применение GSI-I, вероятнее всего, за счёт подавления протеасомной деградации HIF-1.…”