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Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Introduction Tumor immunotherapy have broadened therapeutic options for tumor treatment. The role of immune function in juvenile recurrent respiratory papillomatosis (JRRP) has not been investigated. Applying immunoblockade inhibitors as a novel disease treatment is unclear. Our study, for the first time, evaluates immune infiltration and immuno-suppressive molecule expression in JRRP. Our study provides insights in possibly treating this disease with tumor immunotherapies. We aimed to determine expression of programmed death-ligand 1 (PD-L1), a cancer escape protein, and presence of CD8+ T cell infiltration in tumor microenvironment. Material and methods Seven patients with JRRP (mean age: 7.43; age range 3–17) in this study routinely have their tumors surgical debulked at Massachusetts Eye and Ear Infirmary. Following surgery, samples were de-identified and sent to pathology where they were stained and analyzed. Results Six out of seven patients expressed PD-L1 on tumor cells to various extents. Three patients showed concurrent PD-L1 expression on tumor cells and abundant CD8+ tumor infiltrating lymphocytes as well as PD-L1+ stromal lymphocytes, while PD-L1 expression on tumor cells were not associated with CD8+ tumor infiltrating T cells nor PD-L1+ stromal lymphocytes in the other three patients. HPV 6/11 and p16 was detected in all the patients. There appeared to be no correlation between either PD-L1 expression and CD8+ infiltration and clinical severity as measured by both the number of surgeries per year or Derkay score. Conclusions Despite a small cohort, the expression of p16 and HPV 6/11 in all of the patients confirms the tissues were HPV tumor cells. PD-L1 expression was detected in the vast majority of tumor samples, while inflammatory cell compartments showed a higher degree of variation. Expression of PD-L1 on tumor cells but not inflammatory cells raises the possibility of a tumor cell intrinsic manner of PD-L1 expression. In contrast, a group of patients showed PD-L1 positivity in both tumor and inflammatory cells along with abundant CD8+ tumor infiltrating lymphocytes, suggesting adoptive immune resistance in these tumors and potential benefits from tumor immunotherapy.

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression., Graphical Abstract, Highlights • Amplified MYCN is synthetic lethal with the BCL-2 inhibitor ABT-199 in neuroblastoma • MYCN upregulates the MCL-1 inhibitor, NOXA • MYCN-amplified neuroblastomas are further sensitized to ABT-199 with MLN8237 • ABT-199 with MLN8237 induce tumor regressions in MYCN-amplified neuroblastoma mice, Ham et al. show that MYCN-amplified neuroblastomas are sensitive to treatment with the BCL-2 inhibitor ABT-199 due to MYCN-driven increase of NOXA. Combination treatment with the Aurora Kinase A inhibitor MLN8237 and ABT-199 is synergistic in xenograft models of this tumor type, in part via reducing MCL-1.

Summary Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.

Single cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single cell phenotypes have not been well defined. Combining single cell RNA and protein analytics in pancreatic cancer (PDAC) model systems, we demonstrated the role of stromal fibroblasts in shaping PDAC single cell heterogeneity towards invasive (EMT) and proliferative (PRO) phenotypes. Using highcontent digital imaging of RNA in situ hybridization in 195 tumors, we observed these EMT and PRO subpopulations in 319,626 individual cancer cells. Interestingly, we found these EMT and PRO subpopulations form distinct tumor gland “units” associated with differences in stromal abundance and patient survival. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

CellMinerCDB provides a web-based resource and portal for integrating multiple forms of pharmacological and genomic analyses, unifying the richest cancer cell line datasets (the NCI-60, NCI-SCLC, Sanger/MGH GDSC, and Broad CCLE/CTRP). CellMinerCDB enables data queries for genomics and gene regulatory network analyses, and identifying pharmacogenomic determinants and drug signatures. It leverages overlaps of cell lines and drugs. A panel of 41 drugs was evaluated in parallel in the NCI-60 and GDSC, supporting reproducibility and further pathway analyses across databases. We demonstrate the value of CellMinerCDB for elucidating gene expression determinants, such as methylation and copy number variation; highlight difficulties in assessing mutation burden; the dominant role of SLFN11 for drug response; novel response determinants and genomic signatures for topoisomerase inhibitors and schweinfurthins; drug repositioning for bisacodyl and acetalax for triple negative breast cancer; and the discovery of LIX1L as a novel mesenchymal gene regulating cancer cellular migration and invasiveness.

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.