Targeting the Notch1 and mTOR pathways in a mouse T-ALL model

Abstract

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with ␥-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-endstage Tal1/Ink4a/Arf ϩ/Ϫ leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSItreated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is associated with the misexpression of the basic helix-loop-helix protein TAL1/SCL and LIM-domain only proteins LMO1 and LMO2. [1][2][3][4][5][6][7][8] These oncogenes are found misexpressed in greater than 60% of human T-ALL patients. 3,9 Mouse models of T-ALL recapitulate the disease through ectopic expression of Tal1 in the thymus. [10][11][12] These mice develop respiratory distress due to the presence of large thymic masses and have detectable T-cell blasts in peripheral blood lymphocytes (PBLs), spleen, liver, and kidney. 10 Misexpression of Tal1 results in perturbed thymocyte development by interfering with the basic-helix-loop-helix (bHLH) heterodimer E47/HEB that regulates the expression of genes critical for thymocyte differentiation including Rag1/2, CD4, CD3, and TCR␣/␤. 13,14 Consistent with this finding, loss of the E2A gene that encodes the E47 and E12 bHLH protein is associated with human B-progenitor ALL. 15 Mutations in the Notch1 receptor have been frequently detected in mouse T-ALL models [16][17][18][19] and importantly in 54% of T-ALL patients. 20 These mutations cluster in the heterodimerization domain (HD) 20 and the juxtamembrane (JME) region, 21 or result in truncation of PEST regulatory sequences. 18,20 Mutations in the HD domain result in increased susceptibility to cleavage by the gamma-secretase complex; JME mutations may facilitate metalloprotease cleavage, whereas deletion of PEST regulatory sequences is thought to result in increased Notch1 stability. [21][22][23] Treatment of mouse Tal1 leukemic cell lines in vitro with ␥-secretase inhibitors (GSIs) results in cell cycle arrest and apoptosis, revealing that Notch1 signaling is required for leukemic growth/survival. 18Notch1-mediated leukemic growth in mouse and ...

Acute T-cell leukemias remain dependent on Notch signaling despite PTEN and INK4A/ARF loss

“…Of note, Cullion et al observed murine Tal1 leukemias lacking Pten also to remain sensitive to GSI treatment. 43 These findings raise the question as to what complement of secondary genetic alterations can indeed relieve T-cell leukemias of their addiction to Notch signaling. It was proposed recently that FBW7 mutation may contribute to GSI resistance by reducing c-Myc protein turnover.…”

“…Although early clinical studies have identified goblet cell hyperplasia-associated secretory diarrhea as a doselimiting toxicity, both intermittent dosing strategies 43,48 and coadministration of glucocorticoids 49 may help to ameliorate these gastrointestinal side effects yet allow a therapeutic window to be achieved. We observed GSI resistance to occur in only a small fraction of primary human samples (2/13 assayed; 15%), and we and others 43 have encountered GSI resistance only infrequently in murine leukemias. With respect to the human samples, it is worth noting that all of the primary samples reported here were collected from patients at initial diagnosis.…”

Targeting the PI3K/AKT/mTOR Signaling Axis in Children with Hematologic Malignancies

“…[131,132,[134][135][136][137][138][139][140] Our work and the work of others suggest that rapalogs may have the highest degree of activity against ALL types with the worst prognosis, including pre-T, adult, BCR-ABL and Ikaros mutant. [29,135,141,142] Supporting this are recent data demonstrating that early engraftment of pre-B-ALL in immune-deficient mice is associated with the activation of the mTOR pathway and a very high risk of relapse. [143] Experience with chemotherapy for decades, as well as more recent experience with targeted therapies demonstrates that no single agent is likely to be curative.…”

“…Encouraging results have been found in preclinical studies combining rapalogs with inhibitors of Notch signaling, Jak/stat and the proteasome. [141,147,148] Based on the large amount of preclinical data supporting their use, rapalogs are being investigated in clinical trials in patients with ALL. Two adult patients with ALL have been treated in early-phase trials using single-agent rapalogs.…”

Gamma-secretase inhibitors target tumor-initiating cells in a mouse model of ERBB2 breast cancer

“…The effect of MRK-003 on sphere-and colony-formation by mammary epithelial cell and tumor cell populations To determine whether Notch pathway activity was required for the survival or self-renewal of tumorsphereand mammosphere-forming cells, we used MRK-003, an orally active inhibitor of g-secretase, which is required for the proteolytic processing of Notch receptors (Fan et al, 2006;Konishi et al, 2007;Lewis et al, 2007;Cullion et al, 2009;Rao et al, 2009). We performed quantitative sphere-and colony-forming assays, which can be used to approximate the frequency of mammary epithelial stem and progenitor cells in heterogeneous cell populations (Reynolds and Weiss, 1992;Dontu et al, 2003).…”