Publication Date

2018-11-30

Availability

Embargoed

Embargo Period

2020-11-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2018-11-01

First Committee Member

Noriyuki Kasahara

Second Committee Member

Jonathan H. Schatz

Third Committee Member

Robert B. Levy

Fourth Committee Member

Ramin Shiekhattar

Fifth Committee Member

Lazaros Lekakis

Abstract

The NPM1-ALK fusion kinase drives ALK-positive anaplastic large cell lymphoma (ALK+ ALCL). Mechanisms of NPM1-ALK-driven lymphomagenesis are well defined, but mediators of cancer drug addiction in cells overexpressing it are unknown. Cancer drug addiction arises paradoxically when tumors become dependent on exposure to inhibitors to which they formerly were sensitive. We and others demonstrated previously that addiction to tyrosine kinase inhibitors (TKIs) occurs in resistant ALK+ ALCL over-expressing NPM1-ALK. It is unknown, however, if addiction mechanisms defined recently for solid tumors addicted to MEK/ERK inhibitors apply also to these kinase-driven lymphomas. We consistently found that MEK/ERK activation and other oncogenic pathways downstream from NPM1-ALK, do not mediate TKI addiction. We therefore employed unbiased phosphoproteomics to identify specific candidates, of which STAT1 emerged as the key driver. STAT1 phosphorylation in ALK+ ALCL normally drives its proteasomal degradation, but in TKI-addicted cells withdrawn from drug this mechanism is overwhelmed by massive accumulation of pY701-STAT1. This strongly activates tumor-suppressive STAT1 gene expression, counteracting the STAT3 survival program. We show further that interferon-gamma activation of STAT1 can enhance these effects and also synergizes with ALK TKIs in treatment-naïve ALCL. We therefore define for the first time the specific mechanism of cancer drug addiction in a hematologic malignancy and reveal possible therapeutic strategies to exploit it. To generate an accurate in-vivo model to study the disease, we sought adaptation of CRISPR/Cas9 technology to create a driver fusion oncogene from the endogenous murine loci of transplantable hematopoietic stem cells (HSCs). We employed sgRNAs targeting genomic breakpoints in the Npm1 and Alk genes. This resulted in formation of Npm1-Alk, homologous to the classic human oncogene, in a subset of wild-type HSCs transplanted to recipients. Recipients developed large-cell lymphomas with confirmed presence and expression of Npm1-Alk. T lymphomas were CD30+ polyclonal post-thymic malignancies involving spleen and variably other organs. They were transplantable to secondary animals with shorter latency and showed high sensitivity to ALK-specific kinase inhibitors. These techniques establish proof of principle that CRISPR/Cas9 generation of fusion oncogenes in transplantable HSCs produces hematologic malignancy in mice and are widely adaptable to the modeling of additional lymphomas and leukemias.

Keywords

ALK; ALCL; Resistance; STAT1; CRISPR; Genome-editing

Available for download on Sunday, November 29, 2020

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