Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, through activation of c-Myc. Increased expression of c-Myc induces oncogenic stress that can lead to apoptosis through the Mdm2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases maintain wildtype MDM2 and TP53, we hypothesized that T-ALL cells are selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, although only inducing modest apoptosis in T-ALL cells, resulted in a significant upregulation of pro-apoptotic BH3 domain genes BAX and BBC3. This prompted us to evaluate the combination of idasanutlin with navitoclax, a potent Bcl-2/Bcl-XL inhibitor. Indeed, this combination induced robust synergistic killing of T-ALL PDX lines in vitro. We next examined the in vivo response of four different T-ALL xenografts. Notably, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL. Increased c-Myc expression is associated with increased ribosome biogenesis, which can induce a negative feedback loop, termed the ribosomal (or nucleolar) stress pathway, that inhibits proliferation and induces apoptosis. CDKN2A (encoding ARF and INK4A) and certain sensor ribosomal proteins play key roles in the ribosomal stress pathway and, collectively, are mutated in the majority of T-ALL cases. Based on these data, we hypothesize that mutations in CDKN2A and ribosomal proteins RPL5 and RPL10 contribute to T-ALL pathogenesis by attenuating sensing of c-Myc-induced ribosomal stress. We show that expression of activating NOTCH1 mutations induces ribosomal stress in a primary murine T-cell precursor model. We also show that loss of Cdkn2a results in an expansion of hematopoietic stem cells (HSCs) after induction of proliferative stress by treatment with 5-fluorouracil. Cdkn2a loss also is associated with a marked increase in common lymphoid progenitors (CLP), and preliminary data suggest that CLP self-renewal capacity may be increased in Cdkn2a +/- and Cdkn2a-/- CLPs. Finally, we report progress in model building for the two most frequently mutated ribosomal proteins in T-ALL, RPL5 and RPL10. We have validated successful shRNA based knockdown of RPL5 with an inducible lentiviral system. We also describe a strategy to generate a conditional knock-in mouse model of the R98S point mutation in Ribosomal Protein L10. Taken together, these studies suggest that ribosomal stress may be a specific therapeutic vulnerability in T-ALL and push towards a more complete understanding of this pathway in thymopoiesis. We demonstrate that rescue of apoptotic induction downstream of Notch overactivation, by idasanutlin and navitoclax, is highly active in PDX models. Additionally, we observe that activating Notch mutants may induce ribosomal stress in the cell of origin for T-ALL. We are poised to model ribosomal protein mutations in the T-cell lineage and advance understanding of the ribosomal stress pathway in T-ALL.

Committee Chair

Daniel Link

Committee Members

Hani Zaher; Matthew Walter; Nathan Singh; Timothy Ley

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

3-30-2026

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-7096-8602

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