An integrated model for predicting KRAS dependency

The clinical approvals of KRAS G12C inhibitors have been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency. By integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor's KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters α and λ. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final "K20" model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency. The K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors

Histone deacetylase 11 inhibition promotes breast cancer metastasis from lymph nodes

Lymph node (LN) metastases correspond with a worse prognosis in nearly all cancers, yet the occurrence of cancer spreading from LNs remains controversial. Additionally, the mechanisms explaining how cancers survive and exit LNs are largely unknown. Here, we show that breast cancer patients frequently have LN metastases that closely resemble distant metastases. In addition, using a microsurgical model, we show how LN metastasis development and dissemination is regulated by the expression of a chromatin modifier, histone deacetylase 11 (HDAC11). Genetic and pharmacologic blockade of HDAC11 decreases LN tumor growth, yet substantially increases migration and distant metastasis formation. Collectively, we reveal a mechanism explaining how HDAC11 plasticity promotes breast cancer growth as well as dissemination from LNs and suggest caution with the use of HDAC inhibitors

Tumor-targeted gene therapy with lipid nanoparticles inhibits tumor-associated adipocytes and remodels the immunosuppressive tumor microenvironment in triple-negative breast cancer

Adipocytes are the primary cellular components within the tumor microenvironment (TME) of triple-negative breast cancer (TNBC). Increasing evidence suggests that tumor-associated adipocytes (TAAs) can aggravate tumor progression, exacerbate the immunosuppressive TME and compromise therapeutic efficacy. In this study, the biological effect of TAAs within the breast cancer TME is first investigated, and the C-C Motif Chemokine Ligand 2 (CCL2) which is mainly secreted by TAAs in the extracellular environment is identified as the key mediator. CCL2 recruits immune cells such as monocytes and macrophages that further differentiated into immunosuppressive myeloid-derived suppressor cells (MDSCs) and M2 macrophages. To manipulate CCL2-mediated immune response, a protein trap that binds with CCL2 with high affinity and specificity is designed. The plasmid DNA encoding the CCL2 trap (pCCL2) is specifically delivered to the TME by using targeted lipid-protamine-DNA (LPD) nanoparticles to locally express the CCL2 trap and ameliorate the immunosuppressive TME. Significantly, compared with the commercially available CCL2 antibody, this strategy shows enhanced therapeutic efficacy and appreciable tumor growth inhibition. Furthermore, the pCCL2 trap treatment successfully suppresses TAAs, increases T cell infiltration and decreases the population of immunosuppressive M2 macrophages and MDSCs. Further studies show that the pCCL2 trap could facilitate PD-L1 blockade immunotherapy, demonstrating its translation potential

Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β–induced Serpine1

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKOmice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β–mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1–dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation

Incidence and clinical relevance of non-small cell lung cancer lymph node micro-metastasis detected by staging endobronchial ultrasound-guided transbronchial needle aspiration

Background: Approximately twenty percent of lymph node (LN) negative non-small cell lung cancer (NSCLC) patients who undergo curative intent surgery have pan-cytokeratin immunohistochemistry (IHC)-detectable occult micro-metastases (MMs) in resected LNs. The presence of the MMs in NSCLC is associated worsened outcomes. As a substantial proportion of NSCLC LN staging is conducted using endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), we sought to determine the frequency of detection of occult MMs in EBUS-TBNA specimens and to evaluate the impact of MMs on progression-free and overall survival. Methods: We performed retrospective IHC staining for pan-cytokeratin of EBUS-TBNA specimens previously deemed negative by a cytopathologist based on conventional hematoxylin and eosin staining. The results were correlated with clinical variables, including survival outcomes. Results: Of 887 patients screened, 44 patients were identified meeting inclusion criteria with sufficient additional tissue for testing. With respect to the time of the EBUS-TBNA procedure, 52% of patients were clinical stage I, 34% clinical stage II, and clinical 14% stage IIIa NSCLC. Three patients (6.8%) were found to have cytokeratin positive MMs. All 3 MMs detected were at N2 LN stations. The presence of MMs was associated with significantly decreased progression-free (median 210 vs. 1,293 days, P=0.0093) and overall survival (median 239 vs. 1,120 days, P=0.0357). Conclusions: Occult LN MMs can be detected in EBUS-TBNA specimens obtained during staging examinations and are associated with poor clinical outcomes. If prospectively confirmed, these results have significant implications for EBUS-TBNA specimen analyses and possibly for the NSCLC staging paradigm

Pulmonary Delivery of Nanoparticle-Bound Toll-like Receptor 9 Agonist for the Treatment of Metastatic Lung Cancer

CpG oligodeoxynucleotides are potent toll-like receptor (TLR) 9 agonists and have shown promise as anticancer agents in preclinical studies and clinical trials. Binding of CpG to TLR9 initiates a cascade of innate and adaptive immune responses, beginning with activation of dendritic cells and resulting in a range of secondary effects that include the secretion of pro-inflammatory cytokines, activation of natural killer cells, and expansion of T cell populations. Recent literature suggests that local delivery of CpG in tumors results in superior antitumor effects as compared to systemic delivery. In this study, we utilized PRINT (particle replication in nonwetting templates) nanoparticles as a vehicle to deliver CpG into murine lungs through orotracheal instillations. In two murine orthotopic metastasis models of non-small-cell lung cancer-344SQ (lung adenocarcinoma) and KAL-LN2E1 (lung squamous carcinoma), local delivery of PRINT-CpG into the lungs effectively promoted substantial tumor regression and also limited systemic toxicities associated with soluble CpG. Furthermore, cured mice were completely resistant to tumor rechallenge. Additionally, nanodelivery showed extended retention of CpG within the lungs as well as prolonged elevation of antitumor cytokines in the lungs, but no elevated levels of proinflammatory cytokines in the serum. These results demonstrate that PRINT-CpG is a potent nanoplatform for local treatment of lung cancer that has collateral therapeutic effects on systemic disease and an encouraging toxicity profile and may have the potential to treat lung metastasis of other cancer types

Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival

A circle RNA regulatory axis promotes lung squamous metastasis via CDR1-mediated regulation of golgi trafficking

Lung squamous carcinoma (LUSC) is a highly metastatic disease with a poor prognosis. Using an integrated screening approach, we found that miR-671-5p reduces LUSC metastasis by inhibiting a circular RNA (circRNA), CDR1as. Although the putative function of circRNA is through miRNA sponging, we found that miR-671-5pmore potently silenced an axis of CDR1as and its antisense transcript, cerebellar degeneration related protein 1 (CDR1). Silencing of CDR1as or CDR1 significantly inhibited LUSC metastases and CDR1 was sufficient to promote migration and metastases. CDR1, which directly interacted with adaptor protein 1 (AP1) complex subunits and coatomer protein I (COPI) proteins, no longer promoted migration upon blockade of Golgi trafficking. Therapeutic inhibition of the CDR1as/CDR1 axis with miR-671-5p mimics reduced metastasis in vivo. This report demonstrates a novel role for CDR1 in promoting metastasis and Golgi trafficking. These findings reveal an miRNA/ circRNA axis that regulates LUSC metastases through a previously unstudied protein, CDR1. Significance: This study shows that circRNA, CDR1as, promotes lung squamous migration, metastasis, and Golgi trafficking through its complimentary transcript, CDR1. Significance: This study shows that circRNA, CDR1as, promotes lung squamous migration, metastasis, and Golgi trafficking through its complimentary transcript, CDR1

Combining anti-miR-155 with chemotherapy for the treatment of lung cancers

Purpose The oncogenic miR-155 is upregulated in many human cancers and its expression is increased in more aggressive and therapy resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia and acute lymphoblastic leukemia. Results We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism, and demonstrate that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions Our findings support the existence of a miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death