Activating Notch with a Notch agonist peptide induces apoptosis in AML patient samples.

This report describes an unusual case of cyclin D1 expression by an otherwise typical follicular lymphoma, of low histological grade. BCL2-IGH and CCND1-IGH fusions were identified by interphase fluorescence in situ hybridisation.

Introduction Osteosarcoma (OS) is the most common bone cancer and often has poor outcomes: despite surgery and chemotherapy, only 30% of patients with metastases at diagnosis survive their disease. This stagnant survival curve generates interest in finding novel therapeutic targets and new treatment options to improve outcomes. Tipifarnib (TF) (ZARNESTRA®, R115777) is a small molecule farnesyltransferase inhibitor (FTI). Inhibition of farnesylation causes relocalization of Ras in responsive OS cells, resulting in alternate activation of downstream effectors of Ras in sensitive vs. resistant lines in response to FTI. Markers for susceptibility and pathway(s) affected by FTI in OS need to be determined to identify patients who might benefit from FTI. Methods For Immunofluorescence (IF), OS cells were treated with 1 µM TF or no drug for 24 hours, fixed and stained for pan Ras, and analyzed by standard IF techniques. OS187 cells were treated with 0, 0.01 or 1 µM TF with or without z-vad (a pan caspase inhibitor) for up to 4 days, and cell yield determined daily by automated counting (ViCell). OS cells were treated with 0, 0.01 or 1 µM TF for 24 hours and lysates assessed by western blot for downstream targets of RAS, including MEK, p38 and ERK. OS187 was treated with SB 203580 (p38 MAPK inhibitor), U0126 (MEK inhibitor) TF, or all possible combinations for 72 hours. Cell yield was determined as above. Results OS cells demonstrate variable growth inhibition from FTI, with a 2-log difference between highly responsive (OS187) and highly resistant OS lines (Saos-2). In OS187, response to FTI was associated with redistribution of Ras from a diffuse, perimembranous pattern in untreated cells to a punctuate and perinuclear pattern with FTI as assessed by IF. Neither z-vad, U0126 or SB 203580 rescued cells from FTI-induced growth inhibition induced. Ras activity increased with FTI. Activation of Ras effectors differs between sensitive and resistant cell lines: p38, ERK, MEK, MKK3/6, ATF2 and MSK-1 are activated in sensitive cells in response to FTI. Conclusion Subcellular relocalization of Ras in response to FTI suggests that altering prenylation changes Ras’ interaction with effector molecules. Though we have shown that FTI induces cell death and sub-G1 DNA content in responding cells (Hall et al., 2010), the fact that z-vad does not rescue OS cells from growth inhibition and death suggests that FTI-induced death in OS is caspase independent. While multiple members of the MAPK cascade (especially p38) are activated in response to FTI, neither a p38 nor a MEK inhibitor rescued OS cells from FTI-induced growth inhibition. This suggests that either multiple MAPK components need to be inhibited to rescue cells, or that growth inhibition and cell death are independent of MAPK pathway activation. Regardless of mechanism, the strong response of some OS cells to low concentrations of TF suggests that some patients may benefit from this class of drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5269.

Ras proteins are small GTPases that regulate growth, differentiation, survival, and cell death. Prenylation, the addition of either a farnesyl (15 carbon chain lipid) or geranylgeranyl (20 carbon chain lipid) group, allows Ras and other small GTPases to anchor into the plasma membrane, a necessary step for Ras to activate multiple signaling pathways, including the MAPK cascades. The specific CAAX motif at the C terminus of a protein determines whether it can be farnesylated, geranylgeranylated, or both. There are four highly homologous Ras proteins: K-Ras A, K-Ras B, N-Ras, and H-Ras, each containing a CAAX motif that allows farnesylation. K-Ras B and N-Ras can be alternately geranylgeranylated (GGd) if farnesylation is blocked by a specific small molecule (FTI). Oncogenic Ras mutations are found commonly in cancer, and Ras also can be a major mediator of signals initiated by activated receptor tyrosine kinases (RTKs). Since RTK signaling plays a major role in the biology of sarcomas, we were interested in the mechanisms by which FTIs might affect osteosarcoma cell growth and survival. Osteosarcoma cell lines have variable responses to FTI treatment, including reduced proliferation and programmed cell death at a range of concentrations from 1 nM to 100 nM (Hall et al. 2010). Further, 293T fibroblast cells also have decreased cell viability and increased subG1 and G2/M cell cycle arrest with FTI treatment. 293T cells respond similarly to OS187 our most sensitive osteosarcoma cell line. To assess Ras activity in response to FTI, we co-precipitated activated Ras with the Ras target protein Raf, then probed by western for Ras. Surprisingly, Ras activity was increased in response to FTI in both osteosarcoma cell lines and 293T cells. We also found that the activity of downstream effectors, ERK and p38 MAPK, also increased with FTI (Hall et al, 2010). It was unclear whether the changes in cell growth and survival were due to decrease in farnesylated Ras or the increase in geranylgeranylated Ras. To determine if loss of N-Ras or K-Ras B activity could account for reduced cell yield following FTI, we knocked down each of these proteins in our most responsive osteosarcoma cell line, OS187, and in 293T cells. There was no significant difference in cell yield or cell cycle when either of these proteins was knocked down. To determine if alternate prenylation of N or K-Ras B are responsible for decreased cell yield and/or cell cycle arrest, mutant forms of these proteins will need to be transduced into OS187 and 293T cells for further analysis. In conclusion, FTI caused growth inhibition and cell death for both osteosarcoma cells and 293T cells. FTIs increased Ras activation and the activity of downstream effectors. Since knocking out N or K-Ras B did not reproduce any of these effects, the effects of FTI are not due to the loss of farnesylated Ras. These observations suggest that the effects may be due to increased geranylgeranylated N Ras or K-Ras B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3856. doi:10.1158/1538-7445.AM2011-3856

Background Notch is a well-known oncogene in T-ALL, yet appears to have tumor suppressor effects in B-ALL. These cell type-specific effects of Notch signaling mirror consequences seen in early lymphocyte development and raises the question of how Notch leads to such divergent consequences in closely related cell types. In exploring these Notch mechanisms we discovered a B-ALL specific Notch-mediated reduction in the cell cycle regulator Polo-like kinase-1 (PLK1), revealing a novel targetable kinase in B-ALL. Approach To explore the consequences of Notch-mediated down regulation of cell cycle regulator kinase PLK1, we targeted PLK1 kinase function with the novel PLK1-selective inhibitor poloxin in human B-ALL lines. Results PLK1 is highly expressed in B-ALL verses normal tissues (panel A), correlates with cyclin B expression, is expressed >2-fold higher in B-ALL with t(1;19) than other B-ALL samples, and may predict response of ALL to methotrexate. In our panel of human B-ALL cell lines poloxin induced G2/M growth arrest and decreased cell number by >80% (panel B), and decreased survival in B-ALL cells (>75% AnnexinV+, panel C). PLK1 inhibition led to tumor suppressor p53 stabilization, revealing >5-fold increase in p53 protein levels following poloxin treatment in B-ALL (panel D). Mechanistically, PLK1 inhibition leads to both cytoplasmic re-localization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at Ser20, which destabilizes p53-MDM2 interaction and thus accumulation of p53. Conclusions While exploring the mechanisms of cell type-specific effects of Notch signaling in ALL, we have found a novel therapeutic target, the cell cycle regulator PLK1. Our findings reveal a novel therapeutic approach whereby PLK1-selective inhibition via poloxin induces growth arrest and apoptosis in human B-ALL via consequences on cyclin B and p53 pathways. Disclosures: No relevant conflicts of interest to declare.