Lung cancer remains one of the leading causes for cancer-related death in developed countries. In lung adenocarcinomas, EGFR mutations and EML4-ALK fusions are associated with response to EGFR and ALK inhibition. By contrast, therapeutically exploitable genetic alterations have been lacking in squamous-cell lung cancer. We conducted a systematic search for alterations that are therapeutically amenable and performed high-resolution gene-copy number analyses in a set of 232 lung cancer specimens. We identified frequent and focal FGFR1 amplification in squamous-cell lung cancer (n=155), but not in other lung cancer subtypes, and confirmed its presence in an independent cohort of squamous-cell lung cancer samples employing FISH (22% of cases). Using cell-based screening with the FGFR inhibitor (PD173074) in a large (n=83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth (p=0.0002) and induced apoptosis (p=0.008) specifically in those lung cancer cells carrying amplified FGFR1. We validated the dependency on FGFR1 of FGFR1-amplified cell lines by knockdown of FGFR1 and by ectopic expression of a resistance allele of FGFR1 (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Focal FGFR1 amplification is common in squamous-cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

Purpose: ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown.Experimental Design: By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied.Results: Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity.Conclusions: Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.

Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.

Purpose EML4–ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4–ALK-positive tumors. Experimental Design By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684. Results Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4–ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066. Conclusions Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4–ALK fusions and ALK mutations.

Center Heidelberg) for their excellent technical assistance. We also thank the DKFZ Single-Cell Open Lab (scOpenLab) for the experimental assistance in terms of scRNA-seq. Also, this study was supported by the Heidelberg Center for Personalized Oncology (DKFZ-HIPO). We thank the DKFZ Omics IT and Data Management Core Facility (ODCF) and the DKFZ Genomics and Proteomics Core Facility (GPCF) for their technical support.

SummaryKinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules.

Tumor heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is well-known to affect the efficacy of anti-cancer drugs, most personalized treatment approaches do not account for intratumor heterogeneity. We addressed this issue by studying the heterogeneity of lymph node-derived B cell non-Hodgkin lymphoma (B-NHL) by single cell RNA-sequencing (scRNA-seq) and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subclones and compared their drug response and genomic profiles. Malignant subclones of the same patient responded strikingly different to anti-cancer drugs ex vivo, which recapitulated subclone-specific drug sensitivity during in vivo treatment. Tumor infiltrating T cells represented the majority of non-malignant cells, whose gene expression signatures were similar across all donors, whereas the frequencies of T cell subsets varied significantly between the donors. Our data provide new insights into the heterogeneity of B-NHL and highlight the relevance of intratumor heterogeneity for personalized cancer therapies.