Cor Lieftink

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-β signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-β (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-β results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-β becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-β signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a ‘drug holiday’ and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Intrinsic Resistance to MEK Inhibition in KRAS Mutant Lung and Colon Cancer through Transcriptional Induction of ERBB3

There are no effective therapies for the ~30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment.

RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA) screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA)-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

PTPN11 Is a Central Node in Intrinsic and Acquired Resistance to Targeted Cancer Drugs

Most BRAF (V600E) mutant melanomas are sensitive to selective BRAF inhibitors, but BRAF mutant colon cancers are intrinsically resistant to these drugs because of feedback activation of EGFR. We performed an RNA-interference-based genetic screen in BRAF mutant colon cancer cells to search for phosphatases whose knockdown induces sensitivity to BRAF inhibition. We found that suppression of protein tyrosine phosphatase non-receptor type 11 (PTPN11) confers sensitivity to BRAF inhibitors in colon cancer. Mechanistically, we found that inhibition of PTPN11 blocks signaling from receptor tyrosine kinases (RTKs) to the RAS-MEK-ERK pathway. PTPN11 suppression is lethal to cells that are driven by activated RTKs and prevents acquired resistance to targeted cancer drugs that results from RTK activation. Our findings identify PTPN11 as a drug target to combat both intrinsic and acquired resistance to several targeted cancer drugs. Moreover, activated PTPN11 can serve as a biomarker of drug resistance resulting from RTK activation.

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. Clin Cancer Res; 22(15); 3884–93. ©2016 AACR.

Parallel in vivo and in vitro melanoma RNAi dropout screens reveal synthetic lethality between hypoxia and DNA damage response inhibition.

To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets.

A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

Identification of signalling cascades involved in red blood cell shrinkage and vesiculation

Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

SHP2 is required for growth of KRAS -mutant non-small-cell lung cancer in vivo

RAS mutations are frequent in human cancer, especially in pancreatic, colorectal and non-small-cell lung cancers (NSCLCs)1–3. Inhibition of the RAS oncoproteins has proven difficult4, and attempts to target downstream effectors5–7 have been hampered by the activation of compensatory resistance mechanisms8. It is also well established that KRAS-mutant tumors are insensitive to inhibition of upstream growth factor receptor signaling. Thus, epidermal growth factor receptor antibody therapy is only effective in KRAS wild-type colon cancers9,10. Consistently, inhibition of SHP2 (also known as PTPN11), which links receptor tyrosine kinase signaling to the RAS–RAF–MEK–ERK pathway11,12, was shown to be ineffective in KRAS-mutant or BRAF-mutant cancer cell lines13. Our data also indicate that SHP2 inhibition in KRAS-mutant NSCLC cells under normal cell culture conditions has little effect. By contrast, SHP2 inhibition under growth factor–limiting conditions in vitro results in a senescence response. In vivo, inhibition of SHP2 in KRAS-mutant NSCLC also provokes a senescence response, which is exacerbated by MEK inhibition. Our data identify SHP2 inhibition as an unexpected vulnerability of KRAS-mutant NSCLC cells that remains undetected in cell culture and can be exploited therapeutically.Combined inhibition of SHP2 and MEK is an effective therapeutic approach in non-small-cell lung cancer.

TRIM28 Is an Epigenetic Barrier to Induced Pluripotent Stem Cell Reprogramming

Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large‐scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147–157