Mammalian target of rapamycin (mTOR) represents a key downstream intermediate for a myriad of oncogenic receptor tyrosine kinases. In the case of the insulin-like growth factor (IGF) pathway, the mTOR complex (mTORC1) mediates IGF-1 receptor (IGF-1R)-induced estrogen receptor alpha (ERα) phosphorylation/activation and leads to increased proliferation and growth in breast cancer cells. As a result, the prevalence of mTOR inhibitors combined with hormonal therapy has increased in recent years. Conversely, activated mTORC1 provides negative feedback regulation of IGF signaling via insulin receptor substrate (IRS)-1/2 serine phosphorylation and subsequent proteasomal degradation. Thus, the IGF pathway may provide escape (e.g. de novo or acquired resistance) from mTORC1 inhibitors. It is therefore plausible that combined inhibition of mTORC1 and IGF-1R for select subsets of ER-positive breast cancer patients presents as a viable therapeutic option.

B7-H1 (aka PD-L1) blocking antibodies have been used in treatment of human cancers through blocking B7-H1 expressed by tumor cells; however, their impact on B7-H1 expressing tumor-reactive CD8+ T cells is still unknown. Here, we report that tumor-reactive CD8+ T cells expressing B7-H1 are functional effector cells. In contrast to normal B7-H1 blocking antibody, B7-H1 antibodies capable of activating p38 MAPK lose their antitumor activity by deleting B7-H1+ tumor-reactive CD8+ T cells via p38 MAPK pathway. B7-H1 deficiency or engagement with certain antibody results in more activation of p38 MAPK that leads to T cell apoptosis. DNA-PKcs is a new intracellular partner of B7-H1 in the cytoplasm of activated CD8+ T cells. B7-H1 suppresses p38 MAPK activation by sequestering DNA-PKcs in order to preserve T cell survival. Our findings provide a new mechanism of action of B7-H1 in T cells and have clinical implications in cancer immunotherapy when anti-B7-H1 (PD-L1) antibody is applied.

Focal adhesion kinase (FAK) is overexpressed in many types of tumours, including lung cancer. Y15, a small molecule which inhibits Y397 FAK autophosphorylation, decreases growth of human neuroblastoma, breast and pancreatic cancers. In this study, we investigated the in vitro and in vivo effects of Y15, and the underlying mechanism on non-small cell lung cancer cells.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX) or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID) mice from Epstein-Barr virus (EBV)-infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab) to 5.6% (n = 160 with rituximab), and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

Women diagnosed with cutaneous melanoma have a survival advantage compared to men, which has been hypothesized to be due to difference in behavior and/or biology (sex hormones). It remains controversial whether this advantage is dependent on age or stage of disease. We sought to compare melanoma-specific survival between females in pre, peri, and postmenopausal age groups to males in the same age group, adjusting for stage of disease. This is a retrospective population-based cohort study using the Surveillance, Epidemiology, and End Results (SEER) database. Patients diagnosed from 1 January 1992 through 31 January 2011 with primary invasive cutaneous melanoma were included in our cohort. Melanoma-specific survival was the main outcome studied. Of the 106,511 subjects that were included, 45% were female. Females in all age groups (18-45, 46-54, and ≥55) with localized and regional disease, were less likely to die from melanoma compared to males in the same age group. Among patients with localized and regional disease, the relative risk of death due to melanoma increased with advancing age at diagnosis; this increase was more pronounced among females than males. In contrast, we observed no female survival advantage among patients with distant disease and no effect of age on relative risk of death from melanoma. Females with localized and regional melanoma have a decreased risk of death compared to males within all age groups. Our data show no differences in survival between men and women with metastatic melanoma, indicating that the influence of sex on survival is limited to early stage disease but not confined to pre or perimenopausal age groups.

Tumors acquire numerous mutations during development and progression. When translated into proteins, these mutations give rise to neoantigens that can be recognized by T cells and generate antibodies, representing an exciting direction of cancer immunotherapy. While neoantigens have been reported in many cancer types, the profiling of neoantigens often focused on the class-I subtype that are presented to CD8 + T cells, and the relationship between neoantigen load and clinical outcomes was often inconsistent among cancer types. In this study, we described an informatics workflow, REAL-neo, for identification, quality control (QC), and prioritization of both class-I and class-II human leukocyte antigen (HLA) bound neoantigens that arise from somatic single nucleotide mutations (SNM), small insertions and deletions (INDEL), and gene fusions. We applied REAL-neo to 835 primary breast tumors in the Cancer Genome Atlas (TCGA) and performed comprehensive profiling and characterization of the detected neoantigens. We found recurrent HLA class-I and class-II restricted neoantigens across breast cancer cases, and uncovered associations between neoantigen load and clinical traits. Both class-I and class-II neoantigen loads from SNM and INDEL were found to predict overall survival independent of tumor mutational burden (TMB), breast cancer subtypes, tumor-infiltrating lymphocyte (TIL) levels, tumor stage, and age at diagnosis. Our study highlighted the importance of accurate and comprehensive neoantigen profiling and QC, and is the first to report the predictive value of neoantigen load for overall survival in breast cancer.

Seven different anti-PD-1 and PD-L1 mAbs are now widely used in the United States to treat a variety of cancer types, but no clinical trials have compared them directly. Furthermore, because many of these Abs do not cross-react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by Food and Drug Administration-approved Abs. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor Ag (OVA), followed by subsequent B16-OVA tumor challenge. Surprisingly, both control and Ab-treated mice formed an equally robust OVA-specific T cell response at the time of priming. Despite this, anti-PD-1/L1-treated mice exhibited significantly better tumor rejection versus controls, with avelumab generating the best protection. To determine what could be mediating this, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the avelumab-treated mice, the same phenotype of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of Food and Drug Administration-approved anti-PD-1/L1 mAbs and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for optimizing and better utilizing anti-PD-1/L1 therapeutics.

A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.

Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear. Here we demonstrate that transplanting relatively small numbers of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as to spread cellular senescence to host tissues. Transplanting even fewer senescent cells had the same effect in older recipients and was accompanied by reduced survival, indicating the potency of senescent cells in shortening health- and lifespan. The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, decreased the number of naturally occurring senescent cells and their secretion of frailty-related proinflammatory cytokines in explants of human adipose tissue. Moreover, intermittent oral administration of senolytics to both senescent cell-transplanted young mice and naturally aged mice alleviated physical dysfunction and increased post-treatment survival by 36% while reducing mortality hazard to 65%. Our study provides proof-of-concept evidence that senescent cells can cause physical dysfunction and decreased survival even in young mice, while senolytics can enhance remaining health- and lifespan in old mice.

We describe a phase II clinical trial of the combination of ribociclib and letrozole for treatment of relapsed oestrogen receptor (ER)-positive ovarian cancer (OC) and endometrial cancer (EC). The primary endpoint was the proportion of patients alive, progression-free survival (PFS), and still on treatment at 12 weeks (PFS12), with 45% or greater considered positive.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10(-/-) animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10(-/-) disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11(-/-) animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.

A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

Intraoperative diagnosis is routinely performed on cytology touch preparations (TPs) from core needle biopsies (CNBs). Current interest promotes their utility as an important source of patient tissue for clinical genomic testing. Herein we present whole genome structural variant analysis (SVA) from mate-pair sequencing (MPseq) and whole exome sequencing (WES) mutation calling in DNA directly whole genome amplified (WGA) from TPs. Chromosomal copy changes and somatic DNA junction detection from MPseq of TPs were highly consistent with associated CNBs and bulk resected tissues in all cases. While increased frequency coverage noise from limitations of amplification of limited sample input was significant, this was effectively compensated by natural tumor enrichment during the TP process, which also enhanced variant detection and loss of heterozygosity evaluations from WES. This novel TP methodology enables expanded utility of frequently limited CNB for both clinical and research genomic testing.

Despite recent approval of several new agents, relapsed acute lymphoblastic leukemia (ALL) remains challenging to treat. Sapanisertib (MLN0128/TAK-228) is an oral TORC1/2 inhibitor that exhibited preclinical activity against ALL.

Development of resistance to chemotherapy is a major obstacle in extending the survival of patients with cancer. Although originally defined as an immune checkpoint molecule, B7-H1 (also named as PD-L1 or CD274) was found to play a role in cancer chemoresistance; however, the underlying mechanism of action of B7-H1 in regulation of chemotherapy sensitivity remains unclear in cancer cells. Here we show that development of chemoresistance depends on an increased activation of ERK in cancer cells overexpressing B7-H1. Conversely, B7-H1 knockout (KO) by CRISPR/Cas9 renders human cancer cells susceptible to chemotherapy in a cell-context dependent manner through a reduced activation of p38 MAPK. B7-H1 was found to associate with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and this association promoted or maintained the activation of ERK or p38 MAPK in cancer cells. Importantly, we found that targeting B7-H1 by anti-B7-H1 monoclonal antibody (H1A) increased the sensitivity of human triple negative breast cancer cells to cisplatin therapy in vivo. Our results suggest that targeting B7-H1 by an antibody capable of disrupting B7-H1 signals may be a new approach to sensitize cancer cells to chemotherapy.

CpG oligodeoxynucleotides, as a ligand of toll-like receptor (TLR)-9, have demonstrated promising antitumor effects in some clinical trials; however, its toxicity and low efficacy as a systemic therapy has limited its therapeutic applications. In order to improve its therapeutic efficacy, we investigated the mechanisms of CpG-induced antitumor immunity in the context of CD8+ T cell responses. We show that IL-12 is required for the expansion of IFN-γ producing tumor-reactive CD8+ T cells capable of rejecting tumors. In addition, CpGs reduced PD-1 expression by effector CD8+ T cells via the IL-12 pathway. The combination of CpG and PD-1 blockade show a synergistic effect in generation of systemic antitumor immunity. Our studies define a critical role of IL-12 in CpG-induced antitumor immunity and provide a rationale for combined therapy with TLR agonists and immune checkpoint blockade in cancer treatment.

Lung cancer is the leading cause of cancer-related death worldwide. Here we demonstrate that elevated expression of dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32) and its truncated splice variant t-DARPP promote lung tumor growth, while abrogation of DARPP-32 expression in human non-small cell lung cancer (NSCLC) cells reduces tumor growth in orthotopic mouse models. We observe a novel physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NF-κB2) signaling. Bioinformatics analysis of 513 lung adenocarcinoma patients reveals elevated t-DARPP isoform expression is associated with poor overall survival. Histopathological investigation of 62 human lung adenocarcinoma tissues also shows that t-DARPP expression is elevated with increasing tumor (T) stage. Our data suggest that DARPP-32 isoforms serve as a negative prognostic marker associated with increasing stages of NSCLC and may represent a novel therapeutic target.

Reduced BRCA1 expression causes homologous recombination (HR) repair defects in high-grade serous ovarian cancers (HGSOCs). Here, we demonstrate that BRCA1 is transcriptionally activated by a previously unknown function of ZC3H18. We show that ZC3H18 is a DNA-binding protein that interacts with an E2F site in the BRCA1 promoter where it facilitates recruitment of E2F4 to an adjacent E2F site to promote BRCA1 transcription. Consistent with ZC3H18 role in activating BRCA1 expression, ZC3H18 depletion induces BRCA1 promoter methylation, reduces BRCA1 expression, disrupts HR, and sensitizes cells to DNA crosslinkers and poly(ADP-ribose) polymerase inhibitors. Moreover, in patient-derived xenografts and primary HGSOC tumors, ZC3H18 and E2F4 mRNA levels are positively correlated with BRCA1 mRNA levels, further supporting ZC3H18 role in regulating BRCA1. Given that ZC3H18 lies within 16q24.2, a region with frequent copy number loss in HGSOC, these findings suggest that ZC3H18 copy number losses could contribute to HR defects in HGSOC.