BACKGROUND:Skin cancers, particularly keratinocyte cancers, are the most commonly diagnosed tumors. Although surgery is often effective in early-stage disease, skin tumors are not always easily accessible, can reoccur and have the ability to metastasize. More recently, immunotherapies, including intravenously administered checkpoint inhibitors, have been shown to control some skin cancers, but with off-target toxicities when used in combination. Our study investigated whether peritumoral administration of an antibody combination targeting PD-1, 4-1BB (CD137) and VISTA might control skin tumors and lead to circulating antitumor immunity without off-target toxicity.METHODS:The efficacy of combination immunotherapy administered peritumorally or intravenously was tested using transplantable tumor models injected into mouse ears (primary tumors) or subcutaneously in flank skin (secondary tumors). Changes to the tumor microenvironment were tracked using flow cytometry while tumor-specific, CD8 T cells were identified through enzyme-linked immunospot (ELISPOT) assays. Off-target toxicity of the combination immunotherapy was assessed via serum alanine aminotransferase ELISA and histological analysis of liver sections.RESULTS:The data showed that local administration of antibody therapy eliminated syngeneic murine tumors transplanted in the ear skin at a lower dose than required intravenously, and without measured hepatic toxicity. Tumor elimination was dependent on CD8 T cells and was associated with an increased percentage of CD8 T cells expressing granzyme B, KLRG1 and Eomes, and a decreased population of CD4 T cells including CD4+FoxP3+ cells in the treated tumor microenvironment. Importantly, untreated, distal tumors regressed following antibody treatment of a primary tumor, and immune memory prevented growth of subcutaneous flank tumors administered 50 days after regression of a primary tumor.CONCLUSIONS:Together, these data suggest that peritumoral immunotherapy for skin tumors offers advantages over conventional intravenous delivery, allowing antibody dose sparing, improved safety and inducing long-term systemic memory. Future clinical trials of immunotherapy for primary skin cancer should focus on peritumoral delivery of combinations of immune checkpoint antibodies.

Supplementary Figure S3. Preferential targeting of Side Population (SP) CSCs in NCI-H69 cells by VS-5584.

Supplementary Figure S5. Representative Aldefluor FACS data of cells dissociated from MDA-MB-231 and MCF-7 tumors (Fig. 3B and 3D).

Supplementary Figure Legends. Legends for Supplementary Figures S1-S5

<p>Supplementary Figure S1. Aldefluor+ SUM159 breast cancer cells show enriched CSCs and tumor initiating-capability</p>

<p>Supplementary Materials and Methods. More detailed Materials and Methods</p>

Supplementary Figure S2. Side Population of SUM159 cells enriches for tumor-initiating capability.

Supplementary Figure S4. Representative images of tumorspheres formed by SUM159 and MCF7 cells.

Human papilloma virus (HPV) is responsible for all cases of cervical cancer. While prophylactic vaccines are available, the development of peptide-based vaccines as a therapeutic strategy is still under investigation. In comparison with the traditional and currently used treatment strategies of chemotherapy and surgery, vaccination against HPV is a promising therapeutic option with fewer side effects. A peptide derived from the HPV-16 E7 protein, called 8Qm, in combination with adjuvants showed promise as a therapeutic vaccine. Here, the ability of polymerized natural amino acids to act as a self-adjuvating delivery system as a therapeutic vaccine was investigated for the first time. Thus, 8Qm was conjugated to polyleucine by standard solid-phase peptide synthesis and self-assembled into nanoparticles or incorporated in liposomes. The liposome bearing the 8Qm conjugate significantly increased mice survival and decreased tumor growth after a single immunization. Further, these liposomes eradicated seven-day-old well-established tumors in mice. Dendritic cell (DC)-targeting moieties were introduced to further enhance vaccine efficacy, and the newly designed liposomal vaccine was tested in mice bearing 11-day-old tumors. Interestingly, these DCs-targeting moieties did not significantly improve vaccine efficacy, whereas the simple liposomal formulation of 8Qm-polyleucine conjugate was still effective in tumor eradication. In summary, a peptide-based anticancer vaccine was developed that stimulated strong cellular immune responses without the help of a classical adjuvant.

Immune checkpoint blockade (ICB) is now standard of care for several metastatic epithelial cancers and prolongs life expectancy for a significant fraction of patients. A hostile tumor microenvironment (TME) induced by intrinsic oncogenic signaling induces an immunosuppressive niche that protects the tumor cells, limiting the durability and efficacy of ICB therapies. Addition of receptor tyrosine kinase inhibitors (RTKi) as potential modulators of an unfavorable local immune environment has resulted in moderate life expectancy improvement. Though the combination strategy of ICB and RTKi has shown significantly better results compared to individual treatment, the benefits and adverse events are additive whereas synergy of benefit would be preferable. There is therefore a need to investigate the potential of inhibitors other than RTKs to reduce malignant cell survival while enhancing anti-tumor immunity. In the last five years, preclinical studies have focused on using small molecule inhibitors targeting cell cycle and DNA damage regulators such as CDK4/6, CHK1 and poly ADP ribosyl polymerase (PARP) to selectively kill tumor cells and enhance cytotoxic immune responses. This review provides a comprehensive overview of the available drugs that attenuate immunosuppression and overcome hostile TME that could be used to boost FDA-approved ICB efficacy in the near future.

Immune checkpoint blockade (ICB) has resulted in impressive clinical response rates in the treatment of melanoma and squamous cell carcinoma. Unfortunately, systemic administration of ICB therapy is associated with inconsistent patient responses alongside severe immune-related adverse events (iRAEs), resulting in discontinuation of treatment, especially when anti-PD-1 and anti-CTLA-4 mAb therapies are used in combination. Preclinical studies indicate that locoregional administration of ICB to internal solid tumours enhances immune responses with minimal toxicities, but this approach has not been fully explored in cutaneous tumours.

The development of cancer vaccines has been intensively pursued over the past 50 years with modest success. However, recent advancements in the fields of genetics, molecular biology, biochemistry, and immunology have renewed interest in these immunotherapies and allowed the development of promising cancer vaccine candidates. Numerous clinical trials testing the response evoked by tumour antigens, differing in origin and nature, have shed light on the desirable target characteristics capable of inducing strong tumour-specific non-toxic responses with increased potential to bring clinical benefit to patients. Novel delivery methods, ranging from a patient's autologous dendritic cells to liposome nanoparticles, have exponentially increased the abundance and exposure of the antigenic payloads. Furthermore, growing knowledge of the mechanisms by which tumours evade the immune response has led to new approaches to reverse these roadblocks and to re-invigorate previously suppressed anti-tumour surveillance. The use of new drugs in combination with antigen-based therapies is highly targeted and may represent the future of cancer vaccines. In this review, we address the main antigens and delivery methods used to develop cancer vaccines, their clinical outcomes, and the new directions that the vaccine immunotherapy field is taking.

Non-melanoma skin cancers (NMSC) have a higher incidence than all other cancers combined with cutaneous squamous cell carcinoma (cSCC), capable of metastasis, representing approximately 20% of NMSCs. Given the accessibility of the skin, surgery is frequently employed to treat localized disease, although certain localities, the delineation of clear margins, frequency and recurrence of tumors can make these cancers inoperable in a subset of patients. Other treatment modalities, including cryotherapy, are commonly used for individual lesions, with varying success. Immunotherapy, particularly with checkpoint antibodies, is increasingly a promising therapeutic approach in many cancers, offering the potential advantage of immune memory for protection against lesion recurrence. This review addresses a role for PD-1, 4-1BB and VISTA checkpoint antibodies as monotherapies, or in combination as a therapeutic treatment for both early and late-stage cSCC.

Abstract Effective immunotherapy strategies for cutaneous squamous cell carcinoma (SCC) and its precancerous lesion, actinic keratosis (AK), is an emerging area of interest in skin cancer research. Presently, clinical trials are focusing on systemic administration of immune checkpoint inhibitors and agonistic antibodies in advanced SCC. However, few studies have addressed the intradermal route of administration for antibody therapies, in either early stage SCC or AK. In this study, we examined the effects of intradermal antibody therapy in combination with a topical TLR7/8 agonist, imiquimod, in a mouse model of precancerous skin. Using intradermal administration of anti-4-1BB and anti-PD-1 antibodies in combination with topical administration of imiquimod, precancerous skin grafted onto syngeneic recipients underwent a significant reduction in graft size relative to anti-PD-1/imiquimod and isotype antibody/sorbelene control cream groups. Interestingly, flow cytometry analysis of the anti-4-1bb/PD-1/ imiquimod and isotype/control cream groups, 14 days after cessation of therapy administration, revealed a distinct CD8 T cell subpopulation bearing hallmarks of T cell exhaustion (EOMES, PD-1, TIGIT and Tim-3 positive cells) in the spleen of the treatment group. We conclude that intradermal and topical therapy combinations can induce favourable outcomes in the treatment of skin precancers.

Because cancer stem cells (CSCs) have been implicated in chemo-resistance, metastasis and tumor recurrence, therapeutic targeting of CSCs holds promise to address these clinical challenges to cancer treatment. VS-4718 and VS-6063 are potent inhibitors of focal adhesion kinase (FAK), a non-receptor tyrosine kinase that mediates cell signals transmitted by integrins and growth factor receptors. We report here that inhibition of FAK kinase activity by VS-4718 or VS-6063 preferentially targets CSCs, as demonstrated by a panel of orthogonal CSC assays in cell line models and surgically resected primary breast tumor specimens cultured ex vivo. Oral administration of VS-4718 or VS-6063 to mice bearing xenograft models of triple-negative breast cancer (TNBC) significantly reduced the proportion of CSCs in the tumors, as evidenced by a reduced tumor-initiating capability upon re-implantation in limiting dilutions of cells prepared from these tumors. In contrast, the cytotoxic chemotherapeutic agents, paclitaxel and carboplatin, enriched for CSCs, consistent with previous reports that these cytotoxic agents preferentially target non-CSCs. Importantly, VS-4718 and VS-6063 attenuated the chemotherapy-induced enrichment of CSCs in vitro and delayed tumor regrowth following cessation of chemotherapy. An intriguing crosstalk between FAK and the Wnt/β-catenin pathway was revealed wherein FAK inhibition blocks β-catenin activation by reducing tyrosine 654 phosphorylation of β-catenin. Furthermore, a constitutively active mutant form of β-catenin reversed the preferential targeting of CSCs by FAK inhibition, suggesting that this targeting is mediated, at least in part, through attenuating β-catenin activation. The preferential targeting of cancer stem cells by FAK inhibitors provides a rationale for the clinical development of FAK inhibitors aimed to increase durable responses for cancer patients.

PURPOSE. The purpose of this study was to evaluate mechanisms controlling secretory IgA (SIgA) production, thereby ensuring maintenance of ocular surface health.METHODS. To determine whether the presence of specific gut commensal species regulates SIgA levels and IgA transcripts in the eye-associated lymphoid tissues (EALT), specificpathogen- free (SPF) Swiss Webster (SW) mice were treated with antibiotic cocktails, germfree (GF) SW mice were reconstituted with diverse commensal gut microbiota, or monocolonized with gut-specific commensals. Proteomic profiling and quantitative real-time polymerase chain reaction (qRT-PCR) were used to quantify SIgA and IgA levels. 16S rDNA sequencing was carried out to characterize commensal microbiota.RESULTS. Commensal presence regulated ocular surface SIgA levels and mRNA IgA transcripts in EALT. Oral antibiotic cocktail intake significantly reduced gut commensal presence, while maintaining ocular surface commensal levels reduced SIgA and IgA transcripts in EALT. Analysis of gut microbial communities revealed that SPF SW mice carried abundant Bacteroides organisms when compared to SPF C57BL6/N mice, with B. acidifaciens being the most prominent species in SPF SW mice. Monocolonization of GF SW mice with B. acidifaciens, a strict gut anaerobe, resulted in significant increase of IgA transcripts in the EALT, implying generation of B-cell memory.CONCLUSIONS. These data illustrated a "gut-eye'' axis of immune regulation. Exposure of the host to gut commensal species may serve as a priming signal to generate B-cell repertoires at sites different from the gut, such as EALT, thereby ensuring broad protection.

Cancer stem cells (CSCs) have been implicated in resistance to anticancer therapies, disease recurrence, and metastasis, which represent major challenges in the clinical management of cancer. Therapeutic targeting of CSCs thus holds promise in addressing these challenges. VS-5584 is a selective dual PI3K/mTOR kinase inhibitor that potently inhibits mTORC1, mTORC2 and all four Class I PI3K isoforms. We report here that VS-5584 is up to 30-fold more potent at inhibiting the proliferation and survival of CSCs than non-CSCs in breast cancer cell lines using multiple orthogonal CSC assays. Moreover, VS-5584 preferentially induced apoptosis in Aldefluor-positive CSCs relative to Aldefluor-negative non-CSCs as measured by Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in non-CSCs than CSCs cells. VS-5584 also preferentially diminished CSCs in human breast and small cell lung cancer xenograft models in vivo, as evidenced by marked reduction of tumor-initiating capacity in an in vivo limiting dilution re-implantation assay. Likewise, ex vivo VS-5584 treatment of surgically removed breast and ovarian patient tumors preferentially reduced CSC populations. In contrast, cytotoxic chemotherapeutic agents such as paclitaxel and cisplatin preferentially eliminate non-CSCs and thus enrich the CSC population. Interestingly, preferential targeting of CSCs requires inhibition of multiple components of the PI3K/mTOR pathway; simultaneous knockdown of PI3Kα, PI3Kβ, and mTOR by siRNA phenocopied the effect of VS-5584 and exhibited the strongest preferential targeting of CSCs, while knocking down of individual PI3K isoform or mTOR failed to do so. Consistent with its strong suppression of CSCs, VS-5584 effectively delayed tumor regrowth following chemotherapy in small cell lung cancer xenograft models including a patient-derived primary tumor model. The preferential targeting of CSCs thus prompts a new paradigm for testing VS-5584 in the clinic: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584 at well-tolerated doses and thus enable an optimal therapeutic window. Furthermore, suppression of CSCs by VS-5584 in combination with other agents that target the bulk tumor may represent an appealing strategy to achieve more durable responses for cancer patients. Citation Format: Vihren N. Kolev, Quentin G. Wright, David T. Weaver, Mahesh V. Padval, Jonathan A. Pachter, Qunli Xu. Combined inhibition of PI3K isoforms and mTOR kinase is critical for cancer stem cell inhibition by VS-5584. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A36.

Abstract Cancer stem cells (CSC) have been implicated in disease recurrence, metastasis, and therapeutic resistance, but effective targeting strategies for these cells are still wanting. VS-5584 is a potent and selective dual inhibitor of mTORC1/2 and class I PI 3-kinases. Here, we report that VS-5584 is up to 30-fold more potent in inhibiting the proliferation and survival of CSC compared with non-CSC in solid tumor cell populations. VS-5584 preferentially diminished CSC levels in multiple mouse xenograft models of human cancer, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Likewise, VS-5584 treatment ex vivo preferentially reduced CSC in surgically resected breast and ovarian patient tumors. In contrast, chemotherapeutics such as paclitaxel and cisplatin were less effective in targeting CSC than bulk tumor cells. Mechanistic investigations revealed that preferential targeting of CSC required inhibition of multiple components of the PI3K–mTOR pathway: coordinate RNAi-mediated silencing of PI3Kα, PI3Kβ, and mTOR phenocopied the effect of VS-5584, exhibiting the strongest preferential targeting of CSC, while silencing of individual PI3K isoforms or mTOR failed to replicate the effect of VS-5584. Consistent with CSC ablation, VS-5584 delayed tumor regrowth following chemotherapy in xenograft models of small-cell lung cancer. Taken together, the preferential targeting of CSC prompts a new paradigm for clinical testing of VS-5584: clinical trials designed with CSC-directed endpoints may facilitate demonstration of the therapeutic benefit of VS-5584. We suggest that combining VS-5584 with classic chemotherapy that debulks tumors may engender a more effective strategy to achieve durable remissions in patients with cancer. Cancer Res; 75(2); 446–55. ©2014 AACR.

Abstract Verastem is developing VS-5584, a potent and selective dual inhibitor of the mammalian target of rapamycin complexes 1 and 2 (mTOR) and Class I phosphatidylinositide 3-kinases (PI3K), for the treatment of advanced cancer. The PI3K/mTOR signaling pathway is a key regulator in cancer progression and in the survival of cancer stem cells (CSCs). VS-5584 has been shown to be an equipotent inhibitor of all four human Class I PI3K isoforms and the mTOR kinase, and PI3K signaling has been implicated in the maintenance of CSCs in solid tumors. In multiple orthogonal in vitro assays, VS-5584 has been shown to preferentially target CSCs and exhibited significant antiproliferative activity across multiple cancer cell lines. Furthermore, oral administration of VS-5584 has been shown to reduce CSCs in xenograft models. The in vivo antitumor efficacy of once daily and intermittent oral administration of VS-5584 was evaluated in multiple xenograft tumor models representing small cell (SCLC) and non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC) and mesothelioma. Once daily (QD) treatment with VS-5584 demonstrated potent anti-tumor activity, with mean percentage tumor growth inhibition (TGI) ranging from 40% to 97% (P < 0.05). In these models, TGI was dose-dependent with dosages at and above 15 mg/kg showing good antitumor activity (P < 0.05). Interestingly, tumor regression was observed in 33% (3/9) of mice bearing H69 (SCLC) tumors, 60% (6/10) of mice bearing MDA-MB-468 (TNBC) tumors and 50% (5/10) of mice bearing NCI-H226 mesothelioma tumors. This significant antitumor activity was generally observed at well-tolerated dosages. In studies exploring intermittent dosing schedules, efficacy and tolerability were similar or better with a QD 5 days on/2 days off or Monday, Wednesday, Friday schedule compared to continuous daily dosing. We also explored the efficacy of VS-5584 in combination with either cisplatin or docetaxel. In these studies, VS-5584 plus either cisplatin or docetaxel showed significant TGI compared to cisplatin alone in H69 SCLC and docetaxel alone in A549 NSCLC xenograft models (P < 0.05). This potent in vivo anti-tumor activity in xenograft models of NSCLC, SCLC, TNBC and mesothelioma suggests that VS-5584 has the potential for anticancer activity across a variety of cancer types. Intermittent dosing with VS-5584 was sufficient to achieve good efficacy while minimizing side effects, thus allowing a broader therapeutic window compared to QD dosing. VS-5584 in combination with either cisplatin or docetaxel had enhanced anti-tumor activity compared to either chemotherapeutic agent or VS-5584 alone in two in vivo models of lung cancer. VS-5584 is being evaluated in a Phase 1 clinical trial assessing intermittent dosing in subjects with advanced non-hematologic malignancies or lymphoma. Citation Format: Anthony F. Trombino, Vihren N. Kolev, Quentin G. Wright, Qunli Xu, Mahesh V. Padval. VS-5584 a dual mTORC1/2 and PI3K inhibitor has anti-tumor activity in multiple in vivo xenograft tumor models and enhanced efficacy in combination with cisplatin or docetaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 213. doi:10.1158/1538-7445.AM2014-213