T-ALL represents 10-15% of pediatric and 20-25% of adult cases of acute lymphoblastic leukemia. Activating mutations of NOTCH1 are present in ~60% of cases of T-ALL and are thought to induce leukemic transformation, in part, by activating c-Myc (Myc). Myc induces ribosome biogenesis, which can trigger the nucleolar stress pathway. This is an evolutionarily conserved pathway that includes the translocation of ribosome proteins, RPL5, RPL11, and RPL22 to the nuclear cytoplasm where they bind and alter the activity of p14ARF (ARF) and MDM2, which in turn activates p53, inducing a growth arrest. To overcome this negative feedback loop, over 80% of T- ALL carry biallelic deletions of the CDKN2A (encoding for P16INK4A and ARF). In addition, ~17% of T-ALL carry mutations of RPL5, RPL11, or RPL22. Based on this genetic evidence, we hypothesize that Notch signaling induces nucleolar stress in T-ALL and that inactivation of the nucleolar stress pathway is essential to the molecular pathogenesis of T-ALL. The following specific aims are proposed to test this hypothesis.
Aim 1. To determine whether induction of nucleolar stress during normal T-cell development selects for clones carrying mutations in CDKN2A. Notch signaling plays an essential role in normal early T- cell development. We hypothesize that induction of nucleolar stress during early T-cell development selects for T-cell clones carrying mutations of CDKN2A (or RPL genes). To test this hypothesis, we will isolate T-cell progenitors/precursors from wildtype mice and analyze for signs of nucleolar stress. We also will generate Arf or Ink4a deficient mixed bone marrow chimeras and assess donor chimerism. We predict that nucleolar stress in T-cell progenitors/precursors will result in the selective expansion of Arf deficient cells in the T-cell lineage.
Aim 2. To determine whether activating Notch1 mutations induce nucleolar stress in T cell progenitors. We will assess the impact of the enforced expression of NOTCH1 mutations found in T-ALL on ribosome biogenesis and induction of nucleolar stress using the MOLM13 cell line and primary murine T-cells. In parallel studies, we also will knock-down NOTCH1 expression in several T-ALL xenografts. In each case, nucleolar stress will be assessed
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer for which new therapies are needed. The research proposed here is designed to test the novel hypothesis that cellular stress induced by an increased demand on protein synthesis plays a key role in the development of T-ALL. Agents that target the pathways that mitigate this stress may be selectively toxic to T-ALL cells, suggesting a new type of therapy for T-ALL.
