Mechanisms of multicellular drug resistance and novel approaches for targeted therapy in cancer

Abstract

Dysregulated gene expression, due to genetic and epigenetic aberrations, as well as cancer cell-stroma interactions underlie both tumorigenesis and resistance to anti-cancer therapy. Although governed by common mechanisms, these features are unique to each individual tumor and therefore a personalized treatment regimen needs to be composed for each patient. At the same time, novel therapeutic targets for cancer treatment are warranted to be able to match these individual variations, along with identification of biomarkers for their effective use. STAT3 is a transcriptional regulator involved in both cancer development and therapy resistance. In this thesis we have explored the role of STAT3 as well as interferon-related gene signature in multicellular drug resistance. The second part of this thesis explores the use of novel siRNN prodrugs for silencing of Plk1, a cell cycle kinase, in acute lymphoblastic leukemia (ALL) patient samples.

In Paper I we used multicellular spheroids (MCS) as a model to study genes associated with drug resistance. A subset of interferon-stimulated genes (ISGs), that belong the interferonrelated DNA damage resistance signature (IRDS), was enriched in MCS compared to monolayer culture. We found that a panel of IRDS genes was expressed in cell lines of different origin when grown as MCS or as confluent monolayer culture. The induction of these ISGs depended on increased expression of IRF9 and STAT2. Overexpression of IRF9 alone was sufficient to induce the ISGs and confer resistance to chemotherapeutic agents. In Paper II STAT3 was found to be activated in MCS, downstream of gp130-JAK signaling. STAT3 activity was required for the induced expression of IRF9 and the panel of IRDS genes in MCS. We identified a potential STAT3 binding site in the IRF9 promoter and confirmed that STAT3 was enriched at this site in MCS compared to non-confluent monolayer culture. Together, our data suggest that STAT3 is activated in conditions of high cellular density and drives the transcription of IRF9, which in turn induces the expression of a subset of ISGs that confer resistance to chemotherapeutic drugs.

In Paper III we attempted to identify novel STAT3-interacting proteins that affect transcription of STAT3-target genes. In order to achieve this, we combined chromatin immunoprecipitation using anti-STAT3 antibodies with biotinylation and pull down of DNA, and finally mass spectrometry to identify STAT3 interactors. Among the hits were previously described STAT3-binding proteins, as well as new potential interacting partners. In Paper IV we analyzed the effect of novel self-delivering siRNN prodrugs, targeting cell cycle kinase Plk1, in pediatric ALL. We used CD3/IL-2 to stimulate ALL patient samples in order to induce proliferation and Plk1 expression. Our data demonstrates that the siRNN prodrugs successfully enter cycling ALL cells and induce RNAi mediated knockdown of Plk1, which leads to cell cycle arrest and apoptosis.