Despite recent advances in the use of immunotherapy, only a minority of patients with small cell lung cancer (SCLC) respond to immune checkpoint blockade (ICB). Here, we show that targeting the DNA damage response (DDR) proteins PARP and checkpoint kinase 1 (CHK1) signifi cantly increased protein and surface expression of PD-L1. PARP or CHK1 inhibition remarkably potentiated the antitumor effect of PD-L1 blockade and augmented cytotoxic T-cell infi ltration in multiple immunocompetent SCLC in vivo models. CD8 + T-cell depletion reversed the antitumor effect, demonstrating the role of CD8 + T cells in combined DDR-PD-L1 blockade in SCLC. We further demonstrate that DDR inhibition activated the STING/TBK1/IRF3 innate immune pathway, leading to increased levels of chemokines such as CXCL10 and CCL5 that induced activation and function of cytotoxic T lymphocytes. Knockdown of cGAS and STING successfully reversed the antitumor effect of combined inhibition of DDR and PD-L1. Our results defi ne previously unrecognized innate immune pathway-mediated immunomodulatory functions of DDR proteins and provide a rationale for combining PARP/CHK1 inhibitors and immunotherapies in SCLC. SIGNIFICANCE: Our results defi ne previously unrecognized immunomodulatory functions of DDR inhibitors and suggest that adding PARP or CHK1 inhibitors to ICB may enhance treatment effi cacy in patients with SCLC. Furthermore, our study supports a role of innate immune STING pathway in DDR-mediated antitumor immunity in SCLC.

The Notch signaling pathway mediates cell fate decisions1,2 and is tumor suppressive or oncogenic depending on the context2,3. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine (NE) fate4–6. In small cell lung cancer (SCLC), an aggressive NE lung cancer7, loss-of-function NOTCH mutations and the inhibitory effects of ectopic Notch activation indicate that Notch signaling is tumor suppressive8,9. Here, we show that Notch signaling can be both tumor suppressive and pro-tumorigenic in SCLC. Endogenous activation of the Notch pathway results in a NE to non-NE fate switch in 10-50% of tumor cells in a mouse model of SCLC and in human tumors. This switch is mediated in part by Rest/Nrsf, a transcriptional repressor that inhibits NE gene expression. Non-NE Notch-active SCLC cells are slow growing, consistent with a tumor suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to NE tumor cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumor growth and delays relapse. Thus, SCLC tumors generate their own microenvironment via activation of Notch signaling in a subset of tumor cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select SCLC patients.

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Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology-based and homology-independent processes. Both pathways start the integration process with a site-specific break in the chromosome, typically from a zinc-finger nuclease (ZFN). We previously described an efficient homology-independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene-sized DNA molecules into the chromosomal double-strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology-based integration, are proficient at capture of in vivo-linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN- or TALE nuclease-mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology-driven approaches.

SUMMARY The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb’s tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

Effective targeted therapies for small-cell lung cancer (SCLC), the most aggressive form of lung cancer, remain urgently needed. Here we report evidence of preclinical efficacy evoked by targeting the overexpressed cell-cycle checkpoint kinase CHK1 in SCLC. Our studies employed RNAi-mediated attenuation or pharmacologic blockade with the novel second-generation CHK1 inhibitor prexasertib (LY2606368), currently in clinical trials. In SCLC models in vitro and in vivo, LY2606368 exhibited strong single-agent efficacy, augmented the effects of cisplatin or the PARP inhibitor olaparib, and improved the response of platinum-resistant models. Proteomic analysis identified CHK1 and MYC as top predictive biomarkers of LY2606368 sensitivity, suggesting that CHK1 inhibition may be especially effective in SCLC with MYC amplification or MYC protein overexpression. Our findings provide a preclinical proof of concept supporting the initiation of a clinical efficacy trial in patients with platinum-sensitive or platinum-resistant relapsed SCLC.

Highlights d PKA inhibition or PP2A activation inhibits SCLC growth d The GNAS/PKA axis promotes SCLC growth d Proteomics studies uncover widespread PKA targets and downstream signaling networks d PKA activation promotes a cancer stem cell state

Summary Small cell lung cancer (SCLC) is a neuroendocrine lung cancer characterized by fast growth, early dissemination, and rapid resistance to chemotherapy. We identified a population of long-term tumor-propagating cells (TPCs) in a mouse model of SCLC. This population, marked by high levels of EpCAM and CD24, is also prevalent in human primary SCLC tumors. Murine SCLC TPCs are numerous and highly proliferative but not intrinsically chemoresistant, indicating that not all the clinical features of SCLC are linked to TPCs. SCLC TPCs possess a distinct transcriptional profile compared to non-TPCs, including elevated MYC activity. Genetic and pharmacological inhibition of MYC in SCLC cells to non-TPC levels inhibits long-term propagation but not short-term growth. These studies identify a highly tumorigenic population of SCLC cells in mouse models, cell lines, and patient tumors, and a means to target them in this most fatal form of lung cancer.