The Impact of Nucleolar Stress on Thymocyte Development and T Cell Acute Lymphoblastic Leukemia Transformation
Date of Award
Doctor of Philosophy (PhD)
Increased Notch signaling is thought to be central to T-ALL pathogenesis, with activatingmutations of NOTCH1 in 60% of cases, and Notch pathway activation due to mutations in FBXW7 in an additional 15% of cases. Increased Notch signaling results in markedly increased ribosome biogenesis, inducing a negative feedback loop called the nucleolar stress pathway. This is an evolutionarily conserved homeostatic pathway that is triggered by excessive ribosome biogenesis that serves to limit rRNA synthesis, protein translation, and ultimately cell growth. CDKN2A, encoding both ARF and INK4A, plays a key role in the nucleolar stress pathway. Activation of ARF and INK4A inhibits ribosome biogenesis and induces a p53-dependent growth arrest. Of note, biallelic deletions of CDKN2A are present in the majority (>80%) of T-ALL, and nearly all cases of T-ALL with NOTCH1 mutations. Also relevant are data showing that deletions of CDKN2A are in the founding clone, while mutations in NOTCH1 are often confined to a subclone, suggesting that loss of CDKN2A is an early, potentially driver, event in leukemia transformation, while NOTCH1 mutations are a later event. Together, these observations suggest the hypothesis that increased Notch signaling induces nucleolar stress in T cells and inactivation of the nucleolar stress pathway is essential to the molecular pathogenesis of T-ALL. This hypothesis supports a model in which CDKN2A deletions are an early transformation event that generates a permissive cellular environment for subsequent activating NOTCH1 mutations. To test this hypothesis, we first developed a cell culture system to expand murine double positive T cell progenitors. Progenitor T cells are derived from cytokine stimulated CD117+ fetal liver cells grown in co-culture with OP9 cells expressing delta-like ligand 4. These cells differentiate in culture, giving rise to CD4+ CD8a+ (DP) thymocytes. To test how T-ALL relevant NOTCH1 mutations alter DP thymocytes, we transduced these cells with retrovirus expressing empty vector, wildtype NOTCH1, or activating NOTCH1 mutants (aNOTCH1) carrying hotspot mutations L1594P or L1601P. Increased Notch signaling, as evidence by Hey1 mRNA expression was confirmed in cells expressing aNOTCH1. Consistent with our model, expression of aNOTCH1 induced nucleolar stress, as measured by induction of ARF expression, accumulation of 5’ external transcribed spacer (ETS) containing pre-rRNA, and NPM translocation from nucleoli to the nucleoplasm. Induction of nucleolar stress in aNOTCH1 expressing cells was associated with p53 activation, as measured by increased expression of p53 target genes p21CIP1/WAF1 and BAX. Overall, expression of aNOTCH1 had a negative impact on cellular proliferation with induction of apoptosis. We next explored the impact of aNOTCH1 mutants in vivo, using a retroviral transplantation model. In brief, CD117+ cells were sorted from adult mice and subjected to two rounds of retroviral transduction for NOTCH1 variants. These cells were transplanted into lethally irradiated mice and the contribution of NOTCH (GFP+) expressing cells to hematopoiesis examined over time. The contribution of aNOTCH1 variants to hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs), and T cells was similar to input percentage and was stable over time. In sharp contrast, there was a near complete loss of aNOTCH1 expressing myeloid and B cells. We next examined the impact of CDKN2A loss on HSC maintenance. In CDKN2A+/- or CDKN2A-/- mice the numbers of phenotypic HSCs were similar to wildtype mice. This is expected since the expression of CDKN2A is very low in HSCs under steady state conditions. In contrast, in competitive transplantation assays, both CDKN2A+/- and CDKN2A-/- HSCs consistently outperformed wildtype competitors. We hypothesized that replication stress induced by transplantation may activate CDKN2A expression in WT HSCs, suppressing their proliferation. To test this, CDKN2A+/- or CDKN2A-/- bone marrow chimeras were treated with 5-fluorouracil (5-FU) following stable engraftment. Consistent with our model, treatment with 5-FU resulted in significant (~5-fold) increase in CDKN2A+/- or CDKN2A-/- hematopoietic output. Finally, we modeled the impact of CDKN2A loss on aNOTCH-induced leukemogenesis by retrovirally transducing NOTCH variants into CDKN2A+/- or CDKN2A-/- HSPCs. Compared with wildtype, expression of aNOTCH1 in CDKN2A+/- or CDKN2A-/- HSCs resulted in a marked expansion of HSCs (3.70 ± 0.22-fold) and CLPs (6.95 ± 2.28-fold) but did not rescue the aNOTCH-induced block in myeloid of B lymphoid differentiation. A tumor watch confirmed that CDKN2A loss strongly cooperated with aNOTCH expression to induce T-ALL in mice (incidence of T-ALL at 200 days of 17/17 (100%) in NOTCH1 L1601P CDKN2A-/- cell recipients vs 3/12 (25%) in NOTCH1 L1601P CDKN2A+/+ cell recipients, P<0.0001). In summary, these data provide support for a novel model of T-ALL leukemogenesis in which CDKN2A loss is an early event that provides a fitness advantage under conditions of replication stress that leads to an expanded pool of CDKN2A+/- (or CDKN2A-/-) HSCs and CLPs. Loss of CDKN2A provides a permissive cellular environment for activating NOTCH mutations by attenuating nucleolar stress. These data suggest that targeting the nucleolar stress pathway or ribosome biogenesis may have therapeutic activity in T-ALL.
Chair and Committee
Daniel C Link
Grant A Challen
Krambs, Joseph Ryan, "The Impact of Nucleolar Stress on Thymocyte Development and T Cell Acute Lymphoblastic Leukemia Transformation" (2022). Arts & Sciences Electronic Theses and Dissertations. 2740.
Available for download on Monday, August 19, 2024