Combination immunotherapy including OncoVEXmGMCSF creates a favorable tumor immune micro-environment in transgenic BRAF murine melanoma.

Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma.

Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation.

BRAF is an oncogenic protein kinase that drives cell growth and proliferation through the MEK-ERK signaling pathway. BRAF inhibitors have demonstrated antitumor efficacy in melanoma therapy but have also been found to be associated with the development of cutaneous squamous cell carcinomas (cSCCs) in certain patients. Here, we report that BRAF is phosphorylated at Ser729 by AMP-activated protein kinase (AMPK), a critical energy sensor. This phosphorylation promotes the association of BRAF with 14-3-3 proteins and disrupts its interaction with the KSR1 scaffolding protein, leading to attenuation of the MEK-ERK signaling. We also show that phosphorylation of BRAF by AMPK impairs keratinocyte cell proliferation and cell-cycle progression. Furthermore, AMPK activation attenuates BRAF inhibitor-induced ERK hyperactivation in keratinocytes and epidermal hyperplasia in mouse skin. Our findings reveal a mechanism for regulating BRAF signaling in response to energy stress and suggest a strategy for preventing the development of cSCCs associated with BRAF-targeted therapy.

Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma.

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

Primary cicatricial alopecias are characterized by dysregulation of shared gene expression pathways.

The primary forms of cicatricial (scarring) alopecia (PCA) are a group of inflammatory, irreversible hair loss disorders characterized by immune cell infiltrates targeting hair follicles (HFs). Lichen planopilaris (LPP), frontal fibrosing alopecia (FFA), and centrifugal cicatricial alopecia (CCCA) are among the main subtypes of PCAs. The pathogenesis of the different types of PCAs are poorly understood, and current treatment regimens yield inconsistent and unsatisfactory results. We performed high-throughput RNA-sequencing on scalp biopsies of a large cohort PCA patients to develop gene expression-based signatures, trained into machine-learning-based predictive models and pathways associated with dysregulated gene expression. We performed morphological and cytokine analysis to define the immune cell populations found in PCA subtypes. We identified a common PCA gene signature that was shared between LPP, FFA, and CCCA, which revealed a significant over-representation of mast cell (MC) genes, as well as downregulation of cholesterogenic pathways and upregulation of fibrosis and immune signaling genes. Immunohistological analyses revealed an increased presence of MCs in PCAs lesions. Our gene expression analyses revealed common pathways associated with PCAs, with a strong association with MCs. The indistinguishable differences in gene expression profiles and immune cell signatures between LPP, FFA, and CCCA suggest that similar treatment regimens may be effective in treating these irreversible forms of hair loss.

Improved therapeutic efficacy of unmodified anti-tumor antibodies by immune checkpoint blockade and kinase targeted therapy in mouse models of melanoma.

The use of specific anti-tumor antibodies has transformed the solid cancer therapeutics landscape with the relative successes of therapies such as anti-HER2 in breast cancer, and anti-EGFR in HNSCC and colorectal cancer. However, these therapies result in toxicity and the emergence of resistant tumors. Here, we showed that removing immune suppression and enhancing stimulatory signals increased the anti-tumor activity of unmodified TA99 antibodies (anti-TYRP1) with a significant reduction of growth of solid tumors and lung metastases in mouse models of melanoma. Immune checkpoint blockade enhanced the efficacy of TA99, which was associated with greater CD8+/Foxp3+, NK1.1+ and dendritic cell infiltrates, suggestive of an increased anti-tumor innate and adaptive immune responses. Further, MEK inhibition in melanoma cell lines increased the expression of melanosomal antigens in vitro, and combining TA99 and MEKi in vivo resulted in enhanced tumor control. Moreover, we found an improved therapeutic effect when YUMM tumor-bearing mice were treated with TA99 combined with MEKi and immune checkpoint blockade (anti-PD1 and anti-CTLA4). Our findings suggest that MEKi induced an increased expression of tumor-associated antigens, which in combination with anti-tumor antibodies, generated a robust adaptive anti-tumor response that was sustained by immune checkpoint inhibition therapy. We postulate that combining anti-tumor antibodies with standard-of-care strategies such as immune checkpoint blockade or targeted therapy, will improve therapeutic outcomes in cancer.

Phenformin Promotes Keratinocyte Differentiation via the Calcineurin/NFAT Pathway.

Phenformin is a drug in the biguanide class that was previously used to treat type 2 diabetes. We have reported the antitumor activities of phenformin to enhance the efficacy of BRAF-MAPK kinase-extracellular signal-regulated kinase pathway inhibition and to inhibit myeloid-derived suppressor cells in various melanoma models. Here we demonstrate that phenformin suppresses tumor growth and promotes keratinocyte differentiation in the 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis mouse model. Moreover, phenformin enhances the suspension-induced differentiation of mouse and human keratinocytes. Mechanistically, phenformin induces the nuclear translocation of NFATc1 in keratinocytes in an AMPK-dependent manner. Pharmacologic or genetic inhibition of calcineurin and NFAT signaling reverses the effects of phenformin on keratinocyte differentiation. Taken together, our study reveals an antitumor activity of phenformin to promote keratinocyte differentiation that warrants future translational efforts to repurpose phenformin for the treatment of cutaneous squamous cell carcinomas.

Use of a TGFbeta type I receptor inhibitor in mouse skin carcinogenesis reveals a dual role for TGFbeta signaling in tumor promotion and progression.

Pharmacological inhibitors of the transforming growth factor ß (TGFß) type I receptor (ALK5) have shown promise in blocking growth of xenotransplanted cancer cell lines but the effect on a multistage cancer model is not known. To test this, we treated mouse skin with SB431542 (SB), a well-characterized ALK5 inhibitor, during a two-stage skin carcinogenesis assay. Topical SB significantly reduced the total number, incidence and size of papillomas compared with 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion alone, and this was linked to increased epidermal apoptosis, decreased proliferation and decreased cutaneous inflammation during promotion. In contrast, the frequency of conversion to squamous cell carcinoma (SCC) was 2-fold higher in papillomas treated with SB. Although there was no difference in tumor cell proliferation in early premalignant lesions, those that formed after SB treatment exhibited reduced squamous differentiation and an altered inflammatory microenvironment similar to SCC. In an inducible epidermal RAS transgenic model, treatment with SB enhanced proliferation and cutaneous inflammation in skin but decreased expression of keratin 1 and increased expression of simple epithelial keratin 18, markers of premalignant progression. In agreement with increased frequency of progression in the multistage model, SB treatment resulted in increased tumor formation with a more malignant phenotype following long-term RAS induction. In contrast to the current paradigm for TGFß in carcinogenesis, these results demonstrate that cutaneous TGFß signaling enables promotion of benign tumors but suppresses premalignant progression through context-dependent regulation of epidermal homeostasis and inflammation.

Transforming growth factor beta1 enhances tumor promotion in mouse skin carcinogenesis.

Transforming growth factor beta1 (TGFbeta1) expression is elevated by tumor promoters in the mouse skin, but its role in tumor promotion has not been well defined. To investigate this, we have compared TGFbeta1+/+ and +/- mice in a two-stage skin chemical carcinogenesis protocol. Surprisingly, TGFbeta1+/- mice had fewer number and incidence of benign papillomas, reduced epidermal and tumor cell proliferation and reduced epidermal TGFbeta1 and nuclear p-Smad2 localization in response to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) compared with TGFbeta1+/+ mice. Maximal TPA activation of protein kinase C (PKCalpha) as measured by activity assays and activation of target genes and induction of cornified envelopes correlated with TGFbeta1 gene dosage in keratinocytes and addition of exogenous TGFbeta1 restored the cornification defect in TGFbeta1+/- keratinocytes. Similarly, inhibition of ALK5-suppressed TPA-mediated PKCalpha activation suggesting that physiological levels of TGFbeta1 are required for maximal activation of PKC-dependent mitogenic responses. Paradoxically, the TPA-induced inflammatory response was greater in TGFbeta1+/- skin, but TGFbeta1+/+ papillomas had more tumor infiltrating myeloperoxidase-positive cells and pro-inflammatory gene expression was elevated in v-ras(Ha)-transduced TGFbeta1+/+ but not TGFbeta1+/- keratinocytes. Thus, ras activation switches TGFbeta1 to a pro-inflammatory cytokine. Despite this differential proliferative and inflammatory response to TPA and enhanced papilloma formation in the TGFbeta1+/+ mice, the frequency of malignant conversion was reduced compared with TGFbeta1+/- mice. Therefore, TGFbeta1 promotes benign tumors by modifying tumor promoter-induced cell proliferation and inflammation but retains a suppressive function for malignant conversion.

Context-dependent regulation of cutaneous immunological responses by TGFbeta1 and its role in skin carcinogenesis.

Transforming growth factor beta1 (TGFbeta1) signaling plays a critical role in skin carcinogenesis. While most studies have focused on TGFbeta1 signaling and response in keratinocytes, it is now becoming clear that the interaction of keratinocyte-derived TGFbeta1 with cells of the immune system has an equally important role in tumor development. Tumors form within the context of innate and adaptive immune responses and studies in skin and skin carcinogenesis models have provided important insight into the impact of context-dependent pro-inflammatory and immunosuppressive actions of TGFbeta1 on tumor development. Indeed, the paradigm of TGFbeta1 duality is clearly evident in its ability to both promote and inhibit inflammatory responses. Recent studies have begun to shed new light on the molecular basis for these actions and to provide insight into how these may contribute to context-dependent effects of TGFbeta1 on carcinogenesis in the skin and other epithelial tissues.

IKZF1 Enhances Immune Infiltrate Recruitment in Solid Tumors and Susceptibility to Immunotherapy.

Immunotherapies are some of the most promising emergent treatments for several cancers, yet there remains a majority of patients who do not benefit from them due to immune-resistant tumors. One avenue for enhancing treatment for these patients is by converting these tumors to an immunoreactive state, thereby restoring treatment efficacy. By leveraging regulatory networks we previously characterized in autoimmunity, here we show that overexpression of the master regulator IKZF1 leads to enhanced immune infiltrate recruitment and tumor sensitivity to PD1 and CTLA4 inhibitors in several tumors that normally lack IKZF1 expression. This work provides proof of concept that tumors can be rendered susceptible by hijacking immune cell recruitment signals through molecular master regulators. On a broader scale, this work also demonstrates the feasibility of using computational approaches to drive the discovery of novel molecular mechanisms toward treatment.

Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower Antitumor Therapeutic Responses.

Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors in vitro and in vivo in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. Cancer Res; 77(13); 3513-26. ©2017 AACR.

A tumor suppressor function for the lipid phosphatase INPP4B in melanocytic neoplasms.

The phosphoinositide-3 kinase (PI3K) pathway is deregulated in a significant proportion of melanomas, and PI3K pathway activation in combination with constitutively active mitogen-activated protein kinase signaling shows synergistic effects in the process of melanoma tumorigenesis. Recently, a tumor suppressor function for the lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described in breast and prostate cancers, with impact on PI3K signaling output. Given the importance of PI3K pathway activity for melanoma formation and growth, we aimed to assess the role of INPP4B in melanocytic tumors. Our studies in native tumors suggest that decreased INPP4B expression is an event correlating with tumor progression in melanocytic neoplasms. We further demonstrate that INPP4B regulates PI3K/Akt signaling and exerts a tumor suppressor effect, impacting the proliferative, invasive, and tumorigenic capacity of melanoma cells. INPP4B expression in melanocytic neoplasms may therefore have potential as a biomarker for disease progression and as a modulator for the prediction of treatment outcome.

Targeted inhibition of BRAF kinase: opportunities and challenges for therapeutics in melanoma.

Malignant melanoma is the most aggressive form of skin cancer and its incidence has increased dramatically in the last two decades. Even with a high rate of success in the treatment of early stages of this malignancy, currently there are no effective strategies for the treatment of advanced metastatic melanoma. Much effort has been put into the use of different target-specific drugs, among which BRAF kinase-specific small-molecule inhibitors have rendered promising results as therapeutic agents in metastatic melanoma. Nonetheless, some side effects, such as development of SCC (squamous cell carcinoma), as well as tumour resistance and recurrence, are common limitations of this therapeutic strategy. The use of combination treatments in which different regulatory pathways or the immunological response are targeted seems to be a promising tool for the future success of melanoma therapeutics.

TGFbeta1-induced inflammation in premalignant epidermal squamous lesions requires IL-17.

Overexpression of transforming growth factor-beta1 (TGFbeta1) in the normal epidermis can provoke an inflammatory response, but whether this occurs within a developing tumor is not clear. To test this, we used an inducible transgenic mouse to overexpress TGFbeta1 in premalignant squamous lesions. Within 48 hours of TGFbeta1 induction, there was an increase in IL-17 production by both CD4(+) and gammadelta(+) T cells, together with increased expression of T-helper-17 (Th17)-polarizing cytokines. Induction of TGFbeta1 in premalignant primary keratinocytes elevated the expression of proinflammatory and Th17-polarizing cytokines, and the keratinocyte-conditioned media caused IL-17 production by naive T cells that was dependent on T-cell TGFbeta1 signaling. Microarray analysis showed significant upregulation of proinflammatory genes 2 days after TGFbeta1 induction, and this was followed by increased MPO(+), F4/80(+), and CD8(+) cells in tumors, increased CD8(+) effectors and IFNgamma(+) cells in skin-draining LNs, and tumor regression. In parallel, the percentage of tumor CD11b(+)Ly6G(+) neutrophils was reduced. Neutralization of IL-17 blocked TGFbeta1-induced CD11b(+) Ly6G(-) tumor infiltration but did not alter the reduction of neutrophils or tumor regression. Thus, TGFbeta1 overexpression causes IL-17-dependent and IL-17-independent changes in the premalignant tumor inflammatory microenvironment.

The high-risk benign tumor: evidence from the two-stage skin cancer model and relevance for human cancer.

Benign tumors that form following chemical initiation and promotion in the mouse skin can be grouped into two classes. The majority of papillomas do not progress to squamous cell carcinoma (SCC), and these are designated as low-risk or terminally benign papillomas. In contrast, a much smaller group forms the true precursor to the SCC, and these have a significantly higher frequency and rate of malignant conversion than the bulk of low-risk papillomas. In standard two-stage carcinogenesis studies both tumor types are present, but grossly indistinguishable. Here we describe properties and potential origins of high-risk papillomas and discuss the relevance of this model for certain human cancers with defined premalignant states.

Peripheral blood mononuclear cells proliferation and Th1/Th2 cytokine production in response to streptococcal M protein in psoriatic patients.

BACKGROUND: Psoriasis is a chronic skin disease that is probably a T cell-mediated autoimmune condition which is strongly associated with streptococcal throat infections. Although some groups have associated the involved response with different streptococcal antigens, M protein has been described as the major virulence factor of Streptococcus pyogenes. Thus, it is necessary to describe some features of the cellular responses to this streptococcal antigen. METHODS: Proliferation and Th1/Th2 cytokine production of peripheral blood mononuclear cells (PBMC) in response to total soluble extracts from type M5 S. pyogenes with (TSE37Sp) and without (M(-)TSESp) M protein were analyzed in 10 psoriatic patients and 10 healthy controls. RESULTS: PBMC from both patients and controls proliferated to both extracts. Responses to M(-)TSESp were significantly lower than those to TSE37Sp (P < 0.05). PBMC IL-2 and gammaIFN production after TSE37Sp stimulus was much higher than after M(-)TSESp antigenic stimulation in both groups (P < 0.05). Meanwhile, IL-4 production was quite low in both groups and in response to both extracts. We found a differential production of IL-10 between groups. PBMC from healthy controls responded to TSE37Sp with a much higher production of this cytokine as compared to the responses showed to M(-)TSESp while the cells from psoriatic patients responded without differences in the production of IL-10. CONCLUSION: Results obtained suggest an important Th1 response to M protein in psoriatic patients which could be associated with the cellular responses involved in psoriasis, while healthy subjects respond in a probably non-Th2 IL-10 producing regulatory T cells fashion.

IgG class antibodies to heat shock-induced streptococcal antigens in psoriatic patients.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease. Probably autoimmune in nature, and associated with streptococcal throat infections as a triggering factor. Although many groups have associated the disease with other pathogens, Streptococcus pyogenes seems to be the most important microorganism related to this disease. Therefore, it is necessary to identify the streptococcal antigens involved in the process. METHODS: In this work IgG class antibodies to soluble antigens obtained from Staphyloccus aureus, Candica albicans or S. pyogenes before and after heat shock induction, were analyzed by ELISA in 28 psoriatic patients and 30 healthy donors. RESULTS: In all cases, the patients and the controls had IgG class antibodies to the four antigens. Nevertheless, the IgG levels to the heat shock-induced S. pyognes were statistically different between the patients and the controls (P < 0.001). There was no difference between the groups when the IgG antibodies to the other antigens, including the noninduced streptococcal extract, were analyzed. Additionally, anti-streptolysin O titers and throat cultures were carried out in all patients and controls. No differences between ASO titers were found but the patients were more frequently colonized by pyogenes. CONCLUSION: Results obtained in this study suggest that heat shock-induced proteins from S. pyogenes are associated with psoriasis.

Recognition of Streptococcus pyogenes and skin autoantigens in Guttate psoriasis

Background. Guttate psoriasis is associated with infections by Streptococcus pyogenes and cross-reaction between skin and streptococcal antigens have been reported, suggesting an autoimmune component in the disease. Methods. In this work, the authors looked for antibodies against S. pyogenes M-5 antigens by immunoblot in 52 sera of psoriasis patients and in 52 sera of normal individuals. Histological and immunohistochemical analysis in skin biopsies from lesions of another group of 16 clinically diagnosed guttate psoriasis patients and four healthy controls were also carried out. Results. All guttate psoariasis patients studied (11) had IgG antibodies that intensively recognized three different proteins of 70,60 and 14 kDa, as compared to sera from patients with other forms of psoriasis or from healthy controls. The diagnosis of psoariasis was confirmed in 14 of the patients by hematoxylin-eosin staining. Of the other two patients, one was diagnosed as parapsoriasis and the other as liquen. By indirect immunofluorescence (IFI), all 14 psoriatic patients had autoantibodies against their own lesional skin that did not recognized normal skin from control subjects or from the two non-psoriatic patients. The parapsoriatic and the liquen patients did not have autoantibodies. A rabbit immune serum against S. pyogenes antigens reacted with lesional skin from the 14 guttate psoriatic patients, but not with normal skin from controls or with lesional skin from the 2 non-psoriatic patients. Conclusions. The recognition by immunoblot of streptococcal antigens by serum of guttate psoriasis patients, the presence of autoantibodies against their own skin, and recognition of the same skin antigens by anti-streptococcal rabbit antibodies confirm the participation of the immune system and of streptococcal infection in guttate psoriasis