GSBS Dissertations and Theses

Approval Date

11-8-2016

Document Type

Doctoral Dissertation

Academic Program

Interdisciplinary Graduate Program

Department

Molecular, Cell, and Cancer Biology Department

First Thesis Advisor

Michael R. Green, M.D., Ph.D.

Keywords

Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Antineoplastic Agents, Drug Resistance, Neoplasm, Chronic Myeloid Leukemia

Subjects

Dissertations, UMMS; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Antineoplastic Agents; Drug Resistance, Neoplasm

Abstract

Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive.

First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells.

Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model.

Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients.

Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.

DOI

10.13028/M2HS3V

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Friday, November 17, 2017

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