Date

12-2013

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

Potent and specific kinase inhibitors have revolutionized the treatment of kinase-driven cancers by providing targeted inhibition of known cancer driving kinases. However, disease resistance and disease persistence have prevented targeted kinase therapy from being curative. Depending upon the disease state, kinase inhibitors may be effective for many years or only a few months before secondary resistance develops. Even for diseases where treatment is efficacious for many years, patients must be treated continuously, as interruption of the therapy results in disease growth within a few weeks or months. The goal of this thesis work was to address both disease resistance and persistence in KIT-mutant mast cell cancer models through the development of novel combination therapies. Activating KIT mutations have been found in acute myeloid leukemia (AML), mast cell tumors, melanoma, seminoma, and gastrointestinal stromal tumors (GIST). This thesis focuses on KIT-mutant cell lines derived from mast cell tumors. Mast cell tumors have historically been difficult to treat with targeted kinase inhibitors because 90% of patients have a highly resistant KIT mutation driving their tumors – KIT D816V. The first portion of this thesis is focused on the investigation of a hypothesis that originated from a chronic mylogenous leukemia (CML) model. In the CML model, imatinib was found to synergize with cyclosporine A (CSA) to inhibit the proliferation and survival of BCR-ABL+ CML cell lines. This synergy required inhibition of calcineurin (CN) enzyme activity and hyperphosphorylation of NFAT species. Given similarities in kinase signaling between CML and mast cells with activating KIT mutant alleles, I hypothesized that combining a KIT inhibitor with a CN phosphatase inhibitor such as CSA would synergistically inhibit the growth and survival of KIT-mutant mast cells. To vii characterize the effects of a similar combination on disease resistance in KIT-mutant mast cell lines I measured cell viability, and apoptosis in six distinct KIT-mutant mast cell lines following treatment with a spectrum of KIT and CN inhibitors. I found that combination therapy synergized to reduce cell viability and increase caspase 3/7. To assess the effects of this combination on disease persistence I measured replating efficiency of KIT-mutant P815 cells because of their adherent, colony-forming nature. Here too, after long-term drug exposure, I found that combining CSA with a KIT inhibitor synergized to reduce the replating efficiency of KIT-mutant P815 cells. Next, I investigated the mechanism underlying the observed synergy. Through the use of NFAT-reporter assays, shRNA knockdown of calcineurin, and NFAT specific pharmacologic inhibitors, I was able to determine that the effects of CSA were exerted through inhibition of calcineurin phosphatase activity, and subsequently inhibition of NFAT activity. Notably, NFAT species were found to be constitutively active in all of the KIT-mutant mast cell lines evaluated to date. This could explain the success of combining an NFAT inhibitor with a KIT inhibitor in KIT-mutant mast cells and may suggest a point of crosstalk or cooperation between KIT and NFAT signaling pathways. Following up on my observation that the synergistic effects were at least partially mediated by the transcription factor NFAT, I used an RNA-Seq screen to identify novel combination therapies to test against the CSA plus KIT inhibitor combination in vivo. It was our goal to identify effective combination therapies that would not rely on an immunosuppressive agent (CSA). This approach revealed that following combination therapy in P815 cells, members of the JAK-STAT and “Cancer” pathways were overrepresented in the list of target genes that were significantly downregulated or upregulated. I followed up on this finding by testing three targets within these pathways: JAK1/2, CCND1/2, and MYC. viii Through a sequence of assays (similar to those used to evaluate the CSA plus KIT inhibitor combination) I determined that combination treatments inhibiting both JAK1/2 kinases and KIT kinase with separate inhibitors synergized to the same extent as inhibiting calcineurin plus KIT. Additionally, inhibiting CCND1/2 plus using a CDK4.6 inhibitor synergized with a KIT kinase inhibitor to nearly the same extent, and would also be a good candidate for in vivo testing. In contrast, the MYC plus KIT inhibitor combination did not perform as well as the other combinations with respect to cellular viability, caspase activity, or long term replating efficiency. These results were confirmed with caspase 3/7 assays and target knockdown experiments similar to those performed during the calcineurin investigation. I have concluded that constitutive NFAT signaling may be a characteristic of KIT-mutant mast cells, or perhaps activated mast cells in general, that can be leveraged to selectively sensitize these cells to KIT inhibition. It appears that combining an NFAT inhibitor with a KIT inhibitor synergistically inhibits JAK-STAT signaling leading to cell death and decreased long-term cell viability in KIT-mutant mast cell models. We plan to advance the CSA, JAK, and CCND plus KIT inhibitor combinations to in vivo testing in mice with the hopes of moving these combination therapies into clinical trials.

Identifier

doi:10.6083/M4FB5186

School

School of Medicine

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