Resolution of Melanoma to Programmed Death-1 Blockade but Simultaneous Rapid Progression of Concomitant Chronic Lymphocytic Leukemia.

Here, we present a novel case of a patient with chronic lymphocytic leukemia (CLL) who received CTLA-4 and then PD-1 immune-checkpoint blockade (ICB) as treatment for concomitant metastatic melanoma. Whereas the metastatic melanoma was responsive to ICB, the CLL rapidly progressed (but responded to ICB cessation and ibrutinib). There were no new genetic mutational drivers to explain the altered clinical course. PD-1/PD-L1/PD-L2 and CTLA-4/CD80/CD86 expression was not increased in CLL B cells, CD8+ or CD4+ T-cell subsets, or monocytes. The patient's CLL B cells demonstrated strikingly prolonged in vitro survival during PD-1 blockade, which was not observed in samples taken before or after ICB, or with other patients. To our knowledge, a discordant clinical course to ICB coupled with these biological features has not been reported in a patient with dual malignancies.

Sustained expression of HPV16 E7 oncoprotein promotes p-AKT(Ser473)/p-Src(Tyr527) signaling to drive precancerous lesions to invasive cervical cancer.

Human papillomavirus (HPV) E7 oncogene plays the most important role in cervical cancer. However, whether E7 oncoprotein is continuously expressed, associated with AKT(Ser473)/p-Src(Tyr527) signaling to trigger cervical carcinogenesis remains unclear. Here, we explored first if HPV16 E7 oncoprotein could be detected in clinical biopsies and is sustainedly expressed, and then investigated how this oncoprotein interacted with AKT(Ser473)/p-Src(Tyr527) signaling in cancer progression. We used ZHPV16E7384 affibody to detect E7 expression in HPV16-positive cervical cancer biopsies and animal tumors by immunohistochemistry (IHC). Results showed that ZHPV16E7384 affibody had intense and specific staining for E7 oncoprotein in the detected specimen. The E7 oncoprotein was continuously expressed to correspond with the development of precancerous lesions to invasive cervical cancer. IHC staining also revealed that AKT, p-AKT(Ser473), Src and p-Src(Tyr527) proteins were expressed in both patient biopsies and animal tumors, with the highest levels of p-AKT(Ser473)/p-Src(Tyr527) present in invasive cancer. Furthermore, siRNA experiments revealed that HPV16 E7 knockdown significantly impaired expression of p-AKT(Ser473)/p-Src(Tyr527) in both HPV16 E7-positive cancer cells and transformed cells. In addition, transient expression of HPV16 E7 protein promoted significantly expression of p-AKT(Ser473)/p-Src(Tyr527) in primary human keratinocytes. Finally, co-immunoprecipitation analysis proved that HPV 16 E7 protein interacted reciprocally with p-AKT(Ser473)/p-Src(Tyr527). In conclusion, we demonstrate that HPV16 E7 oncoprotein is continuously expressed to promote expression of p-AKT(Ser473)/p-Src(Tyr527) leading to drive the initiation and progression of cervical cancer. Our data provide a novel insight that HPV16 E7 activates p-AKT(Ser473)/p-Src(Tyr527) to establish a mechanistic link between the oncogene and the AKT/Src signaling to trigger cervical carcinogenesis.

Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

HPV16 E7-impaired keratinocyte differentiation leads to tumorigenesis via cell cycle/pRb/involucrin/spectrin/adducin cascade.

Here, we used codon usage technology to generate two codon-modified human papillomavirus (HPV)16 E7 genes and, together with wild-type E7, to construct three HPV16 E7 gene plasmids: Wt-E7, HB1-E7, and HB2-E7. The three HPV 16 E7 plasmids were used to investigate how HPV16 E7 protein was expressed in different cells and how this oncoprotein deregulated cellular and molecular events in human keratinocytes to induce carcinogenesis. We discovered that codon usage of HPV16 E7 gene played a key role in determining expression of E7 oncoprotein in all tested cells. HPV16 E7 inhibited significantly expression of pRb to impair keratinocyte differentiation and disrupted development of skin epidermis in mice. HPV16 E7 increased substantially the number of G0/G1 cells associated with upregulation of cyclin D2 and downregulation of cyclin B1 in keratinocytes. HPV16 E7 not only inhibited expression of involucrin and α-spectrin but also disrupted the organization of involucrin filaments and spectrin cytoskeleton. Furthermore, HPV16 E7 inhibited expression of ß-adducin, destroyed its cytoskeletal structure and induced phosphorylation of ß-adducin(Ser662) in keratinocytes. Importantly, HPV16 E7 induced carcinogenesis in mice associated with expression of phosphorylated ß-adducin(Ser662) and its nucleus-translocation. In conclusion, we provided evidence that HPV16 E7 oncoprotein inhibited keratinocyte differentiation in vitro and in vivo leading to carcinogenesis through cell cycle arrest and disruption of pRb/involucrin/spectrin/adducin cascade.

SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

Targeted antibody therapies improve outcomes for chronic lymphocytic leukemia (CLL) patients. However, resistance often develops. We have previously shown that resistance to therapeutic antibodies, by monocyte derived macrophages (referred to as nurse like cells, NLCs), from CLL patients is characterized by suppression of antibody dependent phagocytosis (ADP). The mechanism(s) contributing to the muted ADP responses remain unresolved. In this regard, an innate immune checkpoint was recently described that uses the CD47:SIRPα axis to suppress phagocytic responses by macrophages. In this study we examine whether the SIRPα axis regulates ADP responses to the anti-CD20 antibody, obinutuzumab, by NLCs. Using siRNA depletion strategies we show that SIRPα is a suppressor of ADP responses. Moreover, we show that this innate immune checkpoint contributes to the resistance phenotype in NLCs derived from CLL patients. Finally, we show that SIRPα suppression is mediated via the phosphatase, Shp1, which in turn suppresses SYK-dependent activation of ADP. Thus, we identify a druggable pathway that could be exploited to enhance sensitivity to existing therapeutic antibodies used in CLL. This is the first study to show that activation of the CD47:SIRPα innate immune checkpoint contributes to ADP resistance in NLCs from CLL patients.

PI3K-p110δ contributes to antibody responses by macrophages in chronic lymphocytic leukemia.

Fcγ receptor (FcγR) signalling in monocyte derived macrophages from chronic lymphocytic leukaemia (CLL) patients is poorly understood. This signalling pathway is the key determinant of the ability of the macrophages to respond to therapeutic antibodies in current clinical use for CLL. Muted FcγR signalling activity accompanies disease progression and results in resistance to therapeutic antibodies. The molecular mechanisms controlling FcγR signalling and resistance are unknown. Here, we demonstrate that the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is essential for CLL-derived macrophages to respond to therapeutic antibodies. Inhibition of p110δ in the macrophages reduces FcγR-mediated antibody immune responses. Surprisingly, our studies indicated that FcγR downstream signalling is independent of SYK and BTK activity. Thus, we show that FcγR antibody responses occur via a previously unidentified p110δ-dependent pathway, which is independent of the previously described SYK/BTK activation pathway. These data provide novel insights into the effectors of antibody responses. Our data also provide mechanistic insights into therapy resistance in CLL.

Auranofin improves overall survival when combined with standard of care in a pilot study involving dogs with osteosarcoma.

Osteosarcoma is the most common paediatric primary bone malignancy. The major cause of death in osteosarcoma is drug-resistant pulmonary metastasis. Previous studies have shown that thioredoxin reductase 2 is a driver of metastasis in osteosarcoma and can be inhibited by auranofin (AF). Moreover, studies have shown that AF significantly reduces pulmonary metastases in xenotransplant models. Here, we describe a phase I/II study of AF in canine osteosarcoma, a well-recognized spontaneous model of human osteosarcoma. We performed a single-arm multicentre pilot study of AF in combination with standard of care (SOC) (amputation + carboplatin). We recruited 40 dogs to the trial and used a historical SOC-only control group (n = 26). Dogs >15 kg received 9 mg AF q3d PO and dogs <15 kg received 6 mg q3d. Follow-up occurred over at least a 3-year period. Auranofin plus SOC improved overall survival (OS) (P = .036) in all dogs treated. The improved outcome was attributable entirely to improved OS in male dogs (P = .009). At the time of writing, 10 dogs (25%) survive without measurable disease in the treatment group with survival times ranging between 806 and 1525 days. Our study shows that AF improves OS in male dogs when combined with SOC. Our findings have translational relevance for the management of canine and human osteosarcoma. Our data justify a larger multicentre phase 2 trial in dogs and a phase I/II trial in human patients with refractory disease at the time of initial surgery.

An Ex Vivo Human Tumor Assay Shows Distinct Patterns of EGFR Trafficking in Squamous Cell Carcinoma Correlating to Therapeutic Outcomes.

EGFR overexpression is associated with squamous cell carcinoma development. Altered endocytosis and polarization of receptor tyrosine kinases, including EGFR, affect migration and invasion in three-dimensional culture. These studies have been completed via genetic sequencing, cell line, or three-dimensional in vitro and in vivo murine models. Here, we describe an imaging method that allows ex vivo examination of ligand-induced endocytosis of EGFR in non-dissociated human tumors. We analyzed sets of tumor samples from advanced cutaneous squamous cell carcinoma and head and neck squamous cell carcinoma, actinic keratosis, intraepidermal carcinoma, and cutaneous squamous cell carcinoma. We show that EGFR endocytosis is dysregulated in advanced SCC and correlates with anti-EGFR monoclonal antibody therapy outcomes. In actinic keratosis, intraepidermal carcinoma, and well-differentiated cutaneous squamous cell carcinoma, different patterns of epidermal growth factor ligand uptake and binding were observed at the leading edge of different dysplastic lesions, suggesting that these differences in EGFR endocytosis might influence the metastatic potential of dysplastic squamous epithelium. These studies in live ex vivo human tumors confirm that endocytosis dysregulation is a physiological event in human tumors and has therapeutic implications.

Clinically-Relevant Rapamycin Treatment Regimens Enhance CD8+ Effector Memory T Cell Function In The Skin and Allow their Infiltration into Cutaneous Squamous Cell Carcinoma.

Patients receiving immunosuppressive drugs to prevent organ transplant rejection exhibit a greatly increased risk of developing cutaneous squamous cell carcinoma (SCC). However, not all immunosuppressive drugs confer the same risk. Randomised, controlled trials demonstrate that switching renal transplant recipients receiving calcineurin inhibitor-based therapies to mammalian target of rapamycin (mTOR) inhibitors results in a reduced incidence of de novo SSC formation, and can even result in the regression of pre-existing premalignant lesions. However, the contribution played by residual immune function in this setting is unclear. We examined the hypotheses that mTOR inhibitors promote the enhanced differentiation and function of CD8+ memory T cells in the skin. Here, we demonstrate that the long-term oral administration of rapamycin to achieve clinically-relevant whole blood drug target thresholds, creates a "low rapamycin dose" environment in the skin. While both rapamycin and the calcineurin inhibitor tacrolimus elongated the survival of OVA-expressing skin grafts, and inhibited short-term antigen-specific CD8+ T cell responses, rapamycin but not tacrolimus permitted the statistically significant infiltration of CD8+ effector memory T cells into UV-induced SCC lesions. Furthermore, rapamycin uniquely enhanced the number and function of CD8+ effector and central memory T cells in a model of long-term contact hypersensitivity provided that rapamycin was present during the antigen sensitization phase. Thus, our findings suggest that patients switched to mTOR inhibitor regimens likely experience enhanced CD8+ memory T cell function to new antigen-challenges in their skin, which could contribute to their lower risk of de novo SSC formation and regression of pre-existing premalignant lesions.

Targeting the XPO1-dependent nuclear export of E2F7 reverses anthracycline resistance in head and neck squamous cell carcinomas.

Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)-dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer.

Glyco-centric lectin magnetic bead array (LeMBA) - proteomics dataset of human serum samples from healthy, Barrett׳s esophagus and esophageal adenocarcinoma individuals.

This data article describes serum glycoprotein biomarker discovery and qualification datasets generated using lectin magnetic bead array (LeMBA) - mass spectrometry techniques, "Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma" [1]. Serum samples collected from healthy, metaplastic Barrett׳s esophagus (BE) and esophageal adenocarcinoma (EAC) individuals were profiled for glycoprotein subsets via differential lectin binding. The biomarker discovery proteomics dataset consisting of 20 individual lectin pull-downs for 29 serum samples with a spiked-in internal standard chicken ovalbumin protein has been deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the data set identifier PRIDE: PXD002442. Annotated MS/MS spectra for the peptide identifications can be viewed using MS-Viewer (〈http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer〉) using search key "jn7qafftux". The qualification dataset contained 6-lectin pulldown-coupled multiple reaction monitoring-mass spectrometry (MRM-MS) data for 41 protein candidates, from 60 serum samples. This dataset is available as a supplemental files with the original publication [1].

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones.

Osteosarcoma (OS) is the most common pediatric bone tumor and is associated with the emergence of pulmonary metastasis. Unfortunately, the mechanistic basis for metastasis remains unclear. Tumor-derived extracellular vesicles (EVs) have been shown to play critical roles in cell-to-cell communication and metastatic progression in other cancers, but their role in OS has not been explored. We show that EVs secreted by cells derived from a highly metastatic clonal variant of the KHOS cell line can be internalized by a poorly metastatic clonal variant of the same cell line and induce a migratory and invasive phenotype. This horizontal phenotypic transfer is unidirectional and provides evidence that metastatic potential may arise via interclonal co-operation. Proteomic analysis of the EVs secreted by highly metastatic OS clonal variants results in the identification of a number of proteins and G-protein coupled receptor signaling events as potential drivers of OS metastasis and novel therapeutic targets. Finally, multiphoton microscopy with fluorescence lifetime imaging in vivo, demonstrated a preferential seeding of lung tissue by EVs derived from highly metastatic OS clonal variants. Thus, we show that EVs derived from highly metastatic clonal variants of OS may drive metastatic behaviour via interclonal co-operation and preferential colonization of the lungs.

No association between HPV positive breast cancer and expression of human papilloma viral transcripts.

Infectious agents are thought to be responsible for approximately 16% of cancers worldwide, however there are mixed reports in the literature as to the prevalence and potential pathogenicity of viruses in breast cancer. Furthermore, most studies to date have focused primarily on viral DNA rather than the expression of viral transcripts. We screened a large cohort of fresh frozen breast cancer and normal breast tissue specimens collected from patients in Australia for the presence of human papilloma virus (HPV) DNA, with an overall prevalence of HPV of 16% and 10% in malignant and non-malignant tissue respectively. Samples that were positive for HPV DNA by nested PCR were screened by RNA-sequencing for the presence of transcripts of viral origin, using three different bioinformatic pipelines. We did not find any evidence for HPV or other viral transcripts in HPV DNA positive samples. In addition, we also screened publicly available breast RNA-seq data sets for the presence of viral transcripts and did not find any evidence for the expression of viral transcripts (HPV or otherwise) in other data sets. This data suggests that transcription of viral genomes is unlikely to be a significant factor in breast cancer pathogenesis.

Serum Glycoprotein Biomarker Discovery and Qualification Pipeline Reveals Novel Diagnostic Biomarker Candidates for Esophageal Adenocarcinoma.

We report an integrated pipeline for efficient serum glycoprotein biomarker candidate discovery and qualification that may be used to facilitate cancer diagnosis and management. The discovery phase used semi-automated lectin magnetic bead array (LeMBA)-coupled tandem mass spectrometry with a dedicated data-housing and analysis pipeline; GlycoSelector (http://glycoselector.di.uq.edu.au). The qualification phase used lectin magnetic bead array-multiple reaction monitoring-mass spectrometry incorporating an interactive web-interface, Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny), for univariate and multivariate statistical analysis. Relative quantitation was performed by referencing to a spiked-in glycoprotein, chicken ovalbumin. We applied this workflow to identify diagnostic biomarkers for esophageal adenocarcinoma (EAC), a life threatening malignancy with poor prognosis in the advanced setting. EAC develops from metaplastic condition Barrett's esophagus (BE). Currently diagnosis and monitoring of at-risk patients is through endoscopy and biopsy, which is expensive and requires hospital admission. Hence there is a clinical need for a noninvasive diagnostic biomarker of EAC. In total 89 patient samples from healthy controls, and patients with BE or EAC were screened in discovery and qualification stages. Of the 246 glycoforms measured in the qualification stage, 40 glycoforms (as measured by lectin affinity) qualified as candidate serum markers. The top candidate for distinguishing healthy from BE patients' group was Narcissus pseudonarcissus lectin (NPL)-reactive Apolipoprotein B-100 (p value = 0.0231; AUROC = 0.71); BE versus EAC, Aleuria aurantia lectin (AAL)-reactive complement component C9 (p value = 0.0001; AUROC = 0.85); healthy versus EAC, Erythroagglutinin Phaseolus vulgaris (EPHA)-reactive gelsolin (p value = 0.0014; AUROC = 0.80). A panel of 8 glycoforms showed an improved AUROC of 0.94 to discriminate EAC from BE. Two biomarker candidates were independently verified by lectin magnetic bead array-immunoblotting, confirming the validity of the relative quantitation approach. Thus, we have identified candidate biomarkers, which, following large-scale clinical evaluation, can be developed into diagnostic blood tests. A key feature of the pipeline is the potential for rapid translation of the candidate biomarkers to lectin-immunoassays.

Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis.

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

Confluence-Induced Squamous Differentiation Is Not Accompanied by Changes in H3K27me3 Repressive Epigenetic Mark.

Recent studies have reported that epigenetic mechanisms may regulate the initiation and progress of squamous differentiation in normal and transformed keratinocytes. In particular, the role of the repressive H3K27me3 mark in the regulation of squamous differentiation has been prominent. However, there is conflicting literature showing that squamous differentiation may be dependent upon or independent of changes in H3K27me3 status. In this study we have examined the binding of trimethylated H3K27 to the promoters of proliferation or differentiation genes in keratinocytes undergoing squamous differentiation in vitro and in vivo. Initially, we examined the expression levels for EZH1, EZH2, and H3K27me3 in differentiating keratinocytes in vitro and in vivo. We extended this to include H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq). Based on these studies, we could find no evidence for an association between widespread gain or loss of H3K27me3 on the promoters of proliferation-specific or differentiation-specific target genes, respectively, during squamous differentiation in adult human keratinocytes. These data suggest that squamous differentiation may occur independent of regulation by H3K27me3 on proliferation and differentiation genes of normal adult human keratinocytes.

RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent Resistance to Doxorubicin and Is Prognostic for Overall Survival in Squamous Cell Carcinoma.

We have previously shown that E2F7 contributes to drug resistance in head and neck squamous cell carcinoma (HNSCC) cells. Considering that dysregulation of responses to chemotherapy-induced cytotoxicity is one of the major reasons for treatment failure in HNSCC, identifying the downstream effectors that regulate E2F7-dependent sensitivity to chemotherapeutic agents may have direct clinical impact. We used transcriptomic profiling to identify candidate pathways that contribute to E2F7-dependent resistance to doxorubicin. We then manipulated the expression of the candidate pathway using overexpression and knockdown in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and RacGAP1 in a custom tissue microarray (TMA) generated from HNSCC patient samples. Transcriptomic profiling identified RacGAP1 as a potential mediator of E2F7-dependent drug resistance. We validated E2F7-dependent upregulation of RacGAP1 in doxorubicin-insensitive SCC25 cells. Extending this, we found that selective upregulation of RacGAP1 induced doxorubicin resistance in previously sensitive KJDSV40. Similarly, stable knockdown of RacGAP1 in insensitive SCC25 cells induced sensitivity to doxorubicin in vitro and in vivo. RacGAP1 expression was validated in a TMA, and we showed that HNSCCs that overexpress RacGAP1 are associated with a poorer patient overall survival. Furthermore, E2F7-induced doxorubicin resistance was mediated via RacGAP1-dependent activation of AKT. Finally, we show that SCC cells deficient in RacGAP1 grow slower and are sensitized to the cytotoxic actions of doxorubicin in vivo. These findings identify RacGAP1 overexpression as a novel prognostic marker of survival and a potential target to sensitize SCC to doxorubicin.