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

Abstract

NOTCH1 is activated by mutation in more than 50% of human T-cell acute lymphoblastic leukemias (T-ALLs) and inhibition of Notch signaling causes cell-cycle/ growth arrest, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor phosphatase and tensin homolog (PTEN) is also mutated or lost in up to 20% of cases. It was recently observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition, raising concern that patients with PTEN-negative disease may fail Notch inhibitor therapy. As these studies were limited to established cell lines, we addressed this issue using a genetically defined mouse retroviral transduction/bone marrow transplantation model and observed primary murine leukemias to remain dependent on NOTCH1 signaling despite Pten loss, with or without additional deletion of p16 Ink4a /p19 Arf IntroductionThe 4 mammalian Notch genes (NOTCH1-4) encode a family of highly conserved type I transmembrane receptors that are normally activated by ligands of the Delta/Serrate/Lag-2 family expressed on the surface of neighboring cells. Once activated by ligand, the Notch receptors undergo proteolytic cleavage first by a disintegrin and metalloprotease, then by a ␥-secretase, which releases the intracellular domain (ICN) from the plasma membrane to translocate to the nucleus to stimulate transcription of downstream target genes in complex with the DNA-binding factor CBF1(RBPJ)/ suppressor of hairless/Lag-1 and coactivators of the Mastermind family. Although regulated NOTCH1 signaling is important for normal T-cell development, 1 it is frequently activated by mutation in the human cancer T-cell acute lymphoblastic leukemia (T-ALL). 2 The potent oncogenicity of activated NOTCH1 has been demonstrated in murine bone marrow transduction/transplantation models and several transgenic mouse lines. 3 Activating NOTCH1 mutations occur in more than 50% of primary human T-ALLs and cluster in the heterodimerization (HD) and C-terminal proline-, glutamic acid-, serine-, and threonine-rich (PEST) domains. 4 HD mutations result in weakened association or complete dissociation of the receptor subunits, and thus lead to heightened/constitutive activation of the receptor. 5 PEST domain mutations often generate premature stop codons that delete the PEST degron, and thus enhance signaling by reducing turnover/ prolonging half-life of activated ICN. 6 When present together, the HD and PEST mutations occur in cis, and stimulate signaling in a synergistic fashion. 4 Interestingly, a similar overall frequency of Notch1 mutations (mostly PEST, but some HD) has been observed in various mouse models of T-ALL, underscoring the importance of Notch1 signaling in T-cell leukemogenesis. In addition, both human and murine T-ALL cells bearing NOTCH1 mutations are frequently sensitive to treatment with inhibitors of Notch signaling including ␥-secretase inhibitors (GSIs) that induce G1 cell-cycle/ growth arrest and in some cases apoptosis. 4,7-10 Based on these findings, GSIs and other inhi...

High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling

“…High titer, replication-defective retroviral supernatants were produced by transient transfection of PlatE cells as described previously (Medyouf et al, 2010). MSCV-based retroviral expression vectors included IRES-GFP cassettes for fluorescent tagging of transduced cells.…”

“…Primary samples were obtained at initial diagnosis with informed consent from patients or their legal guardians or as discarded pathological material under approved Institutional Review Board protocols at the Karmanos Cancer Institute, Hôpital Armand-Trousseau, Hopital Saint-Louis, BC Cancer Agency, and BC Children's and Women's Hospital following guidelines established by the Declaration of Helsinki. Expansion of primary human T-ALL cells in irradiated NOD-Scid/IL2Rc / mice has been described previously (Medyouf et al, 2010).…”

“…PI3K-Akt pathway activation occurs in >85% of cases (Silva et al, 2008) via diverse mechanisms, including mutation or inactivation of PTEN (Kawamura et al, 1999;Perentesis et al, 2004;Maser et al, 2007;Palomero et al, 2007;Silva et al, 2008;Gutierrez et al, 2009) and mutation of PIK3 and Akt (Kawamura et al, 1999;Gutierrez et al, 2009). Activation of PI3K-Akt has been shown to collaborate with Notch in leukemogenesis (Medyouf et al, 2010), enhance growth of established leukemias (Chiarini et al, 2009;Cullion et al, 2009;Levy et al, 2009;Sanda et al, 2010), and in some contexts to samples Weng et al, 2006;Medyouf et al, 2010). For mouse cells, we examined primary leukemias derived by retroviral transduction/transplantation of bone marrow with an activated form of NOTCH1 termed E (Pear et al, 1996).…”

Synergistic Effects of Interleukin-7 and Pre-T Cell Receptor Signaling in Human T Cell Development

“…5A). Molt3 is a ␥-secretase inhibitor-resistant cell line and has PTEN deficiency and therefore has constitutively active Akt (60). We found that pre-TCR signaling enhanced Akt activation on some occasions, but the effect lacked statistical significance.…”

p53 as a Therapeutic Target in T-ALL

“…Inhibition of PI3K, AKT and mTOR kinases was also suggested (Chiarini et al, 2010;Chiarini et al, 2009;Evangelisti et al, 2011a;Evangelisti et al, 2011b). However, despite a significant overlap in downstream targets, PTEN loss cannot compensate for NOTCH1 oncogenic function (Medyouf et al, 2010). Thus inhibition of both pathways was shown to cooperate in primary leukemic T cells and in mouse tumor models (Guo et al, 2011;Cullion et al, 2009).…”