Intrinsic resistance to PIM kinase inhibition in AML through p38α-mediated feedback activation of mTOR signaling

Although conventional therapies for acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) are effective in inducing remission, many patients relapse upon treatment. Hence, there is an urgent need for novel therapies. PIM kinases are often overexpressed in AML and DLBCL and are therefore an attractive therapeutic target. However, in vitro experiments have demonstrated that intrinsic resistance to PIM inhibition is common. It is therefore likely that only a minority of patients will benefit from single agent PIM inhibitor treatment. In this study, we performed an shRNA-based genetic screen to identify kinases whose suppression is synergistic with PIM inhibition. Here, we report that suppression of p38α (MAPK14) is synthetic lethal with the PIM kinase inhibitor AZD1208. PIM inhibition elevates reactive oxygen species (ROS) levels, which subsequently activates p38α and downstream AKT/mTOR signaling. We found that p38α inhibitors sensitize hematological tumor cell lines to AZD1208 treatment in vitro and in vivo. These results were validated in ex vivo patient-derived AML cells. Our findings provide mechanistic and translational evidence supporting the rationale to test a combination of p38α and PIM inhibitors in clinical trials for AML and DLBCL

Improving cancer therapy through unraveling drug resistance

The discovery that the growth and/or survival of cancer cells is dependent on certain oncogenic drivers — also described as ‘oncogene addiction’ — provided a rationale for the development of targeted agents that specifically inhibit aberrant oncoproteins. Unfortunately, the initial expectation that precision medicine would revolutionize cancer treatment turned out to be overly optimistic. The success of targeted cancer therapies is hampered by (1) a lack of predictive biomarkers, (2) the rapid development of therapy resistance, and (3) a paucity of suitable drug targets. The work described in this thesis aimed to provide insights that address these problems. In chapter 2 we describe how resistance to PIM kinase inhibitors in acute myeloid leukemia (AML) occurs through p38α-mediated feedback activation of mTOR signaling. We provide a rationale for combining PIM and p38 inhibitors to overcome therapy resistance. In chapter 3 we identify PIM kinases as a potential therapeutic target and prognostic biomarker in neuroblastoma. We furthermore demonstrate that NF1 loss mediates resistance to PIM kinase inhibitors and is a potential predictive biomarker for PIM inhibitor therapy. In chapter 4 we identify FGFR1 as a potential prognostic biomarker in head and neck squamous cell carcinoma (HNSCC). We suggest that FGFR inhibitors might be useful for the treatment of HNSCC — either as monotherapy or in combination with an EGFR inhibitor for FGFR inhibitor-resistant HNSCC. In chapter 5 we discuss the role of TGF-β signaling as a drug resistance mechanism to both targeted and conventional therapies. We demonstrate that elevated TGF-β signaling is associated with a poor prognosis subtype in colorectal cancer and that TGF-β might induce chemotherapy resistance by suppressing apoptosis. In chapter 6 I discuss the challenges that need to be overcome to improve the benefit of targeted therapies

RIG-I-like receptors and intracellular Toll-like receptors in antiviral immunity

Viral recognition by pattern recognition receptors is a crucial step in antiviral immunity. Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) represent two classes of nucleic acid-sensing pattern recognition receptors that play a major role in inducing an antiviral response. Whereas nucleic acid-recognizing TLRs are transmembrane receptors localized in endosomes, RLRs are distributed within the cytoplasm. Recognition of viral nucleic acid by either class of receptors results in activation of downstream signaling pathways. This eventually induces expression of type I IFN and inflammatory cytokines via activation of the transcription factors IRF3, NF-kappa B and AP-1. Many viruses, such as the extensively studied family of Paramyxoviridae, have evolved sophisticated mechanisms to evade these responses. This review focuses on the differences between viral recognition, signaling pathways and induction of adaptive immunity evoked by RLRs and intracellular TLR

FGFR1 is a potential prognostic biomarker and therapeutic target in head and neck squamous cell carcinoma

Purpose: FGFR1 is a promising therapeutic target in multiple types of solid tumors, including head and neck squamous cell carcinoma (HNSCC). FGFR inhibitors have shown great therapeutic value in preclinical models. However, resistance remains a major setback. In this study, we have investigated the prognostic value of FGFR1 expression in HNSCC, the therapeutic relevance of targeting FGFR with AZD4547, and potential resistant mechanisms. Experimental Design: IHC and FISH were applied on tissue microarrays to investigate FGFR1 protein expression and FGFR1 gene copy numbers in 452 HNSCCs. The sensitivity of HNSCC cell lines to AZD4547, either as single or combination treatment with the EGFR inhibitor gefitinib, was assessed using long-term colony formation assays, short-term viability assays, and biochemical analysis. Results: FGFR1 protein overexpression occurred in 82% (36/ 44) of human papillomavirus (HPV)-positive HNSCC and 75% (294/392) of HPV-negative HNSCC and relates with poor overall survival and disease-free survival in HPV-negative HNSCC [HR, 3.07; 95% confidence interval (CI), 1.74-6.90; P = 0.001 and HR, 1.53; 95% CI, 1.04-2.39; P = 0.033]. Moreover, the FGFR1 gene was amplified in 3% (3/110) of HPV-negative HNSCC. Treatment of the high FGFR1-expressing cell line CCL30 with AZD4547 reduced cell proliferation and FGFR signaling. Two FGFR-amplified cell lines, SCC147 and BICR16, were resistant to AZD4547 treatment due to EGFR signaling. Combined AZD4547 and gefitinib treatment synergistically inhibited the proliferation of resistant cell lines. Conclusions: Here, we identify high FGFR1 expression as a candidate prognostic biomarker in HPV-negative HNSCC. Furthermore, we provide a rationale for treating FGFR1-expressing HNSCC with the FGFR inhibitor AZD4547 and for combining AZD4547 and gefitinib in FGFR inhibitor-resistant HNSCC patients

Nuclear association of Foxp3 and SIRT1.

<p>(<b>A</b>) HEK293 cells were co-transfected with both HA-Foxp3 and Flag-SIRT1, lysed, and Foxp3 was immunoprecipitated and association of proteins was analyzed by Western blotting utilizing anti-Flag antibodies. (<b>B</b>) HA-Foxp3 and Flag-SIRT1 transfected cells were lysed, SIRT1 was immunoprecipitated and the association of Foxp3 was assessed as in (<b>A</b>). (<b>C</b>) SIRT1-Foxp3 interaction in transfected HEK293 cells was visualized using <i>in situ</i> proximity ligation assay (PLA). Cells were fixed and protein-protein interactions were visualized utilizing anti-HA and anti-Flag antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019047#s2" target="_blank">Materials and Methods</a> section. Punctate staining (red) indicates Foxp3-SIRT1 interaction as detected by the assay. Nuclei were stained using Hoechst. Representative images from at least three independent experiments are depicted.</p