Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Dr. Robert Lewis

Abstract

Multiple studies have revealed that Ras-driven tumors acquire vulnerabilities by adapting cellular mechanisms that promote uncontrolled proliferation and suppress apoptosis. Kinase Suppressor of Ras 1 (KSR1) modulates ERK activation downstream of oncogenic Ras, and knockdown of KSR1 selectively kills malignant, Ras-driven cancer cells, but does not kill immortalized, non-transformed human colon epithelial cells (HCECs). KSR1-/- mice are fertile and phenotypically normal, but resistant to Ras-driven tumor formation suggesting KSR1 represents a vulnerability in cancer cells.

To identify additional vulnerabilities in cancer, a screening approach termed Functional Signature Ontology (FUSION) was used to screen 14,355 genes and 1,200 natural product fractions in K-Ras-mutant HCT116 colon cancer cells for functional similarity to KSR1 and a selective requirement in colon cancer cells. FUSION identified numerous targets including TIMELESS, WDR5, and an AMPK inhibitor, 5’-hydroxy-staurosporine.

Downstream of oncogenic signaling, TIMELESS is constitutively overexpressed in multiple types of cancer and required for increased cancer cell proliferation. TIMELESS depletion increases γH2AX, a marker of DNA damage, and triggers downstream G2/M arrest via increased CHK1 and CDK1 phosphorylation. Wee1 or CHK1 inhibition in combination with TIMELESS depletion demonstrates at least additive effects suggesting this combination may be efficacious for the treatment of cancer.

WDR5 is overexpressed, and WDR5 depletion reduces cell viability in colon cancer cells by reducing H3K4Me3 and increasing γH2AX, which further sensitizes cells to radiation-induced DNA damage. WDR5 inhibition also reduces colon cancer cell viability, but less so than WDR5 depletion.

The catalytic, kinase-containing a2 subunit isoform of AMPK is expressed at variable levels in colon cancer cells and is selectively required for colon cancer cell survival suggesting that AMPK kinase inhibition may be a useful component of cancer therapeutic strategies. FUSION identified 5´-hydroxy-staurosporine as a competitive inhibitor of AMPK that is selectively toxic to colon cancer cells.

Our results demonstrate the ability of FUSION to reveal functional similarities between genes, identify novel inhibitors, and expose oncogene-induced changes in cancer that promote proliferation and survival, but may also leave cancer cells vulnerable to targeted therapies.

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