Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition.

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Development of AMOEBA Polarizable Force Field for Rare-Earth La3+ Interaction with Bioinspired Ligands.

Rare-earth metals (REMs) are crucial for many important industries, such as power generation and storage, in addition to cancer treatment and medical imaging. One promising new REM refinement approach involves mimicking the highly selective and efficient binding of REMs observed in relatively recently discovered proteins. However, realizing any such bioinspired approach requires an understanding of the biological recognition mechanisms. Here, we developed a new classical polarizable force field based on the AMOEBA framework for modeling a lanthanum ion (La3+) interacting with water, acetate, and acetamide, which have been found to coordinate the ion in proteins. The parameters were derived by comparing to high-level ab initio quantum mechanical (QM) calculations that include relativistic effects. The AMOEBA model, with advanced atomic multipoles and electronic polarization, is successful in capturing both the QM distance-dependent La3+-ligand interaction energies and experimental hydration free energy. A new scheme for pairwise polarization damping (POLPAIR) was developed to describe the polarization energy in La3+ interactions with both charged and neutral ligands. Simulations of La3+ in water showed water coordination numbers and ion-water distances consistent with previous experimental and theoretical findings. Water residence time analysis revealed both fast and slow kinetics in water exchange around the ion. This new model will allow investigation of fully solvated lanthanum ion-protein systems using GPU-accelerated dynamics simulations to gain insights on binding selectivity, which may be applied to the design of synthetic analogues.

Integrated slice-specific dynamic shimming diffusion weighted imaging (DWI) for rectal Cancer detection and characterization.

PURPOSE: To compare integrated slice-specific dynamic shimming (iShim) diffusion weighted imaging (DWI) and single-shot echo-planar imaging (SS-EPI) DWI in image quality and pathological characterization of rectal cancer. MATERIALS AND METHODS: A total of 193 consecutive rectal tumor patients were enrolled for retrospective analysis. Among them, 101 patients underwent iShim-DWI (b = 0, 800, and 1600 s/mm2) and 92 patients underwent SS-EPI-DWI (b = 0, and 1000 s/mm2). Qualitative analyses of both DWI techniques was performed by two independent readers; including adequate fat suppression, the presence of artifacts and image quality. Quantitative analysis was performed by calculating standard deviation (SD) of the gluteus maximus, signal intensity (SI) of lesion and residual normal rectal wall, apparent diffusion coefficient (ADC) values (generated by b values of 0, 800 and 1600 s/mm2 for iShim-DWI, and by b values of 0 and 1000 s/mm2 for SS-EPI-DWI) and image quality parameters, such as signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of primary rectal tumor. For the primary rectal cancer, two pathological groups were divided according to pathological results: Group 1 (well-differentiated) and Group 2 (poorly differentiated). Statistical analyses were performed with p < 0.05 as significant difference. RESULTS: Compared with SS-EPI-DWI, significantly higher scores of image quality were obtained in iShim-DWI cases (P < 0.001). The SDbackground was significantly reduced on b = 1600 s/mm2 images and ADC maps of iShim-DWI. Both SNR and CNR of b = 800 s/mm2 and b = 1600 s/mm2 images in iShim-DWI were higher than those of b = 1000 s/mm2 images in SS-EPI-DWI. In primary rectal cancer of iShim-DWI cohort, SIlesion was significantly higher than SIrectum in both b = 800 and 1600 s/mm2 images. ADC values were significantly lower in Group 2 (0.732 ± 0.08) × 10- 3 mm2/s) than those in Group 1 ((0.912 ± 0.21) × 10- 3 mm2/s). ROC analyses showed significance of ADC values and SIlesion between the two groups. CONCLUSION: iShim-DWI with b values of 0, 800 and 1600 s/mm2 is a promising technique of high image quality in rectal tumor imaging, and has potential ability to differentiate rectal cancer from normal wall and predicting pathological characterization.

MicroRNA-874-3p promotes testosterone-induced granulosa cell apoptosis by suppressing HDAC1-mediated p53 deacetylation.

MicroRNA (miR)-874-3p is a newly identified miRNA that is involved in several pathological processes, including cancer, myocardial infarction, bone formation and erectile dysfunction. However, the role of miR-874-3p in polycystic ovary syndrome (PCOS) and granulosa cell (GC) apoptosis is not completely understood. The present study investigated the expression profile of miR-874-3p in PCOS by reverse transcription- quantitative PCR and the GC apoptosis by flow cytometry analysis. miR-874-3p expression was significantly upregulated in GCs isolated from patients with PCOS compared with patients without PCOS. In addition, miR-874-3p expression was positively correlated with GC apoptosis and testosterone levels in both patients with PCOS and patients without PCOS. Therefore, the present study also aimed to investigate the effects of miR-874-3p on testosterone-induced GC apoptosis. Compared with vehicle-treated GCs, miR-874-3p expression levels were significantly increased in testosterone-treated GCs, which was inhibited by the androgen receptor antagonist flutamide. GCs were transfected with either the miR-874-3p mimic or a miR-874-3p inhibitor. Compared with the control group, miR-874-3p mimic significantly enhanced GC apoptosis, whereas miR-874-3p inhibitor significantly decreased GC apoptosis. Moreover, histone deacetylase (HDAC) activity and HDAC1 expression levels were decreased in testosterone-treated GCs compared with vehicle-treated GCs. HDAC1 overexpression significantly attenuated the proapoptotic effects of testosterone. Additionally, miR-874-3p mimic and inhibitor significantly decreased and increased HDAC1 expression levels, respectively, compared with the control group. miR-874-3p inhibitor failed to attenuate HDAC1 overexpression-induced GC apoptosis. Furthermore, compared with the control group, testosterone treatment notably increased p53 expression and acetylation. Compared with the control group, western blotting analysis showed that miR-874-3p mimic notably increased p53 expression and acetylation, whereas miR-874-3p inhibitor markedly decreased p53 expression and acetylation. However, miR-874-3p inhibitor did not further decrease p53 acetylation and expression in cell overexpressing HDAC1. Collectively, the results of the present study indicated that miR-874-3p was upregulated in PCOS and promoted testosterone-induced GC apoptosis by suppressing HDAC1-mediated p53 deacetylation. Therefore, the present study improved the current understanding of the pathogenesis of PCOS and GC apoptosis.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

miR-200 affects tamoxifen resistance in breast cancer cells through regulation of MYB.

Resistance to tamoxifen is a major clinical challenge. Research in recent years has identified epigenetic changes as mediated by dysregulated miRNAs that can possibly play a role in resistance to tamoxifen in breast cancer patients expressing estrogen receptor (ER). We report here elevated levels of EMT markers (vimentin and ZEB1/2) and reduced levels of EMT-regulating miR-200 (miR-200b and miR-200c) in ER-positive breast cancer cells, MCF-7, that were resistant to tamoxifen, in contrast with the naïve parental MCF-7 cells that were sensitive to tamoxifen. Further, we established regulation of c-MYB by miR-200 in our experimental model. C-MYB was up-regulated in tamoxifen resistant cells and its silencing significantly decreased resistance to tamoxifen and the EMT markers. Forced over-expression of miR-200b/c reduced c-MYB whereas reduced expression of miR-200b/c resulted in increased c-MYB We further confirmed the results in other ER-positive breast cancer cells T47D cells where forced over-expression of c-MYB resulted in induction of EMT and significantly increased resistance to tamoxifen. Thus, we identify a novel mechanism of tamoxifen resistance in breast tumor microenvironment that involves miR-200-MYB signaling.

The Effect of Topoisomerase I Inhibitors on the Efficacy of T-Cell-Based Cancer Immunotherapy.

Background: Immunotherapy has increasingly become a staple in cancer treatment. However, substantial limitations in the durability of response highlight the need for more rational therapeutic combinations. The aim of this study is to investigate how to make tumor cells more sensitive to T-cell-based cancer immunotherapy. Methods: Two pairs of melanoma patient-derived tumor cell lines and their autologous tumor-infiltrating lymphocytes were utilized in a high-throughput screen of 850 compounds to identify bioactive agents that could be used in combinatorial strategies to improve T-cell-mediated killing of tumor cells. RNAi, overexpression, and gene expression analyses were utilized to identify the mechanism underlying the effect of Topoisomerase I (Top1) inhibitors on T-cell-mediated killing. Using a syngeneic mouse model (n = 5 per group), the antitumor efficacy of the combination of a clinically relevant Top1 inhibitor, liposomal irinotecan (MM-398), with immune checkpoint inhibitors was also assessed. All statistical tests were two-sided. Results: We found that Top1 inhibitors increased the sensitivity of patient-derived melanoma cell lines (n = 7) to T-cell-mediated cytotoxicity (P < .001, Dunnett's test). This enhancement is mediated by TP53INP1, whose overexpression increased the susceptibility of melanoma cell lines to T-cell cytotoxicity (2549 cell line: P = .009, unpaired t test), whereas its knockdown impeded T-cell killing of Top1 inhibitor-treated melanoma cells (2549 cell line: P < .001, unpaired t test). In vivo, greater tumor control was achieved with MM-398 in combination with α-PD-L1 or α-PD1 (P < .001, Tukey's test). Prolonged survival was also observed in tumor-bearing mice treated with MM-398 in combination with α-PD-L1 (P = .002, log-rank test) or α-PD1 (P = .008, log-rank test). Conclusions: We demonstrated that Top1 inhibitors can improve the antitumor efficacy of cancer immunotherapy, thus providing the basis for developing novel strategies using Top1 inhibitors to augment the efficacy of immunotherapy.

HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes.

T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.Many patients fail to respond to T cell based immunotherapies. Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.

Synthetic Poly(L-Glutamic Acid)-conjugated CpG Exhibits Antitumor Efficacy With Increased Retention in Tumor and Draining Lymph Nodes After Intratumoral Injection in a Mouse Model of Melanoma.

There is an urgent need for new clinically applicable drug-delivery methods to enhance accumulation of immune-activating drugs in tumors. We synthesized a poly(L-glutamic acid)-CpG ODN2216 conjugate (PG-CpG) and injected it intratumorally into C57BL/6 mice bearing subcutaneous B16-ovalbumin melanoma. PG-CpG elicited the same potent antitumoral activity as CpG with respect to reducing tumor growth and triggering antigen-specific CD8 T-cell responses in this well-established solid tumor model. Moreover, PG-CpG was retained significantly longer in both tumor and draining lymph nodes than was free CpG after intratumoral injection. Specifically, 48 hours after injection, 26.5%±16.9% of the injected PG-CpG dose versus 4.72%±2.61% of free CpG remained at the tumor, and 1.53%±1.22% of the injected PG-CpG versus 0.37%±0.33% of free CpG was retained in the draining inguinal lymph nodes. These findings indicate that PG is an effective synthetic polymeric carrier for delivery of immunostimulatory agents to tumors and lymph nodes.

A mitochondrial membrane protein is a target for rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV; Reoviridae) is exclusively transmitted by the brown planthopper Nilaparvata lugens in a persistent-propagative manner. It is understood that RNA viral proliferation is associated with the intracellular membranes of the insect host cells. However, the molecular mechanisms of the interaction between the RRSV proliferation and the intracellular membranes remain essentially unknown. It will be of great interest to determine whether RRSV protein(s) directly interact with intracellular membrane components of its host cells. In this study, we identified a RRSV nonstructural protein Pns10 interacting with a host oligomycin-sensitivity conferral protein (OSCP) using yeast two-hybrid system. The interaction between RRSV Pns10 and N. lugens OSCP was verified by a glutathione S-transferase pull-down assay. Confocal miscopy revealed colocalization of these two proteins in the cytoplasm of the salivary gland cells during the viral infection. The virions were further detected in the mitochondria under confocal miscopy and transmission electron microscopy combined with western blotting assay. This is the first observation that RRSV protein has a direct link with mitochondria. Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects. These findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane and provide insights concerning the mitochondrial membrane protein-based virus proliferation mode in the insect vector.

IL2 Variant Circumvents ICOS+ Regulatory T-cell Expansion and Promotes NK Cell Activation.

Clinical responses to high-dose IL2 therapy are limited due to selective expansion of CD4+CD25+Foxp3+ T-regulatory cells (Treg), especially ICOS+ Tregs, rather than natural killer (NK) cells and effector T cells. These ICOS+ Tregs are highly suppressive and constitutively express high levels of IL2Rα (CD25) and CD39. Here, we characterized the effect of a mutant form of IL2 (F42K), which preferentially binds to the lower affinity IL2Rßγ with reduced binding to CD25, on Tregs, effector NK cells, and T-cell subsets. Unlike wild-type (WT) IL2, F42K did not efficiently induce the expansion of highly suppressive ICOS+ Tregs in peripheral blood mononuclear cells (PBMC) from healthy controls and melanoma patients. Instead, it promoted the expansion of CD16+CD56+ NK cells and CD56hiCD16- NK cell subsets in both short- and long-term cultures, with enhanced Bcl-2 expression. Stimulation of PBMCs with F42K induced expression of more NK cell activation molecules, such as NKp30, NKp44, DNAM-1, NKG2D, 4-1BB/CD137, and Tim-3, than WT IL2. F42K induced greater upregulation of TRAIL, and NK-mediated cytolytic activity was increased against both autologous and HLA-mismatched melanoma cells compared with WT IL2. Gene expression analysis revealed distinct gene expression profiles stimulated by F42K, WT IL2, and IL15. F42K therapy in vivo also induced a dramatic reduction in the expansion of ICOS+ Tregs, promoted NK cell expansion, and inhibited melanoma tumor growth more efficiently than WT IL2 and more effectively than anti-CTLA-4. Our findings suggest that F42K could be a potential substitute for WT IL2 as a cytokine therapy for cancer. Cancer Immunol Res; 4(11); 983-94. ©2016 AACR.

Bypassing STAT3-mediated inhibition of the transcriptional regulator ID2 improves the antitumor efficacy of dendritic cells.

Despite the potent ability of dendritic cells (DCs) to stimulate lymphocyte responses and host immunity, granulocyte-macrophage colony-stimulating factor-derived DCs (GM-DCs) used as antitumor vaccines have demonstrated relatively modest success in cancer immunotherapy. We found that injecting GM-DCs into melanoma tumors in mice, or culturing GM-DCs with melanoma-secreted cytokines or melanoma-conditioned medium, rapidly suppressed DC-intrinsic expression of the gene encoding inhibitor of differentiation 2 (ID2), a transcriptional regulator. Melanoma-associated cytokines repressed Id2 transcription in murine DCs through the activation of signal transducer and activator of transcription 3 (STAT3). Enforced expression of ID2 in GM-DCs (ID2-GM-DCs) suppressed their production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Vaccination with ID2-GM-DCs slowed the progression of melanoma tumors and enhanced animal survival, which was associated with an increased abundance of tumor-infiltrating interferon-γ-positive CD4(+) effector and CD8(+) cytotoxic T cells and a decreased number of tumor-infiltrating regulatory CD4(+) T cells. The efficacy of the ID2-GM-DC vaccine was improved by combinatorial treatment with a blocking antibody to programmed cell death protein-1 (PD-1), a current immunotherapy that overcomes suppressive immune checkpoint signaling. Collectively, our data reveal a previously unrecognized STAT3-mediated immunosuppressive mechanism in DCs and indicate that DC-intrinsic ID2 promotes tumor immunity by modulating tumor-associated CD4(+) T cell responses. Thus, inhibiting STAT3 or overexpressing ID2 selectively in DCs may improve the efficiency of DC vaccines in cancer therapy.

Hypoxia-Driven Mechanism of Vemurafenib Resistance in Melanoma.

Melanoma is molecularly and structurally heterogeneous, with some tumor cells existing under hypoxic conditions. Our cell growth assays showed that under controlled hypoxic conditions, BRAF(V600E) melanoma cells rapidly became resistant to vemurafenib. By employing both a three-dimensional (3D) spheroid model and a two-dimensional (2D) hypoxic culture system to model hypoxia in vivo, we identified upregulation of HGF/MET signaling as a major mechanism associated with vemurafenib resistance as compared with 2D standard tissue culture in ambient air. We further confirmed that the upregulation of HGF/MET signaling was evident in drug-resistant melanoma patient tissues and mouse xenografts. Pharmacologic inhibition of the c-Met/Akt pathway restored the sensitivity of melanoma spheroids or 2D hypoxic cultures to vemurafenib. Mol Cancer Ther; 15(10); 2442-54. ©2016 AACR.

Loss of PTEN Promotes Resistance to T Cell-Mediated Immunotherapy.

UNLABELLED: T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kß inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors. SIGNIFICANCE: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K-AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K-AKT pathway to increase the efficacy of immunotherapy.

Persistent antigen at vaccination sites induces tumor-specific CD8⁺ T cell sequestration, dysfunction and deletion.

To understand why cancer vaccine-induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund's adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8(+) T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.

BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma.

PURPOSE: To evaluate the effects of BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. EXPERIMENTAL DESIGN: Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10 to 14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib) and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T-cell markers, and immunomodulatory cytokines. RESULTS: Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T-cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines [interleukin (IL)-6 and IL-8] and an increase in markers of T-cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 was increased on treatment. A decrease in melanoma antigen expression and CD8 T-cell infiltrate was noted at time of progression on BRAF inhibitor alone and was reversed with combined BRAF and MEK inhibition. CONCLUSIONS: Together, these data suggest that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T-cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T-cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma.

BRAF inhibition increases tumor infiltration by T cells and enhances the antitumor activity of adoptive immunotherapy in mice.

PURPOSE: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. EXPERIMENTAL DESIGN: BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2D(b) to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. RESULTS: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model. CONCLUSION: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell-based immunotherapy for the treatment of patients with melanoma.

Antitumor T-cell responses contribute to the effects of dasatinib on c-KIT mutant murine mastocytoma and are potentiated by anti-OX40.

Targeted and immune-based therapies are thought to eradicate cancer cells by different mechanisms, and these approaches could possibly complement each other when used in combination. In this study, we report that the in vivo antitumor effects of the c-KIT inhibitor, dasatinib, on the c-KIT mutant P815 mastocytoma tumor were substantially dependent on T cell-mediated immunity. We found that dasatinib treatment significantly decreased levels of Tregs while specifically enhancing tumor antigen-specific T-cell responses. We sought to further enhance this therapy with the addition of anti-OX40 antibody, which is known to provide a potent costimulatory signal to T cells. The combination of dasatinib and anti-OX40 antibody resulted in substantially better therapeutic efficacy compared with either drug alone, and this was associated with enhanced accumulation of tumor antigen-specific T cells in the tumor microenvironment. Furthermore, the combination regimen inhibited the function of Tregs and also resulted in significantly up-regulated expression of the IFN-γ-induced chemokines CXCL9, 10, and 11 in the tumor microenvironment, which provides a feasible mechanism for the enhanced intratumoral CTL infiltration. These studies delineate a strategy by which targeted therapy and immunotherapy may be combined to achieve superior antitumor responses in cancer patients.

PD-1 blockade enhances T-cell migration to tumors by elevating IFN-γ inducible chemokines.

Adoptive cell transfer (ACT) is considered a promising modality for cancer treatment, but despite ongoing improvements, many patients do not experience clinical benefits. The tumor microenvironment is an important limiting factor in immunotherapy that has not been addressed fully in ACT treatments. In this study, we report that upregualtion of the immunosuppressive receptor programmed cell death-1 (PD-1) expressed on transferred T cells at the tumor site, in a murine model of ACT, compared with its expression on transferred T cells present in the peripheral blood and spleen. As PD-1 can attenuate T-cell-mediated antitumor responses, we tested whether its blockade with an anti-PD-1 antibody could enhance the antitumor activity of ACT in this model. Cotreatment with both agents increased the number of transferred T cells at the tumor site and also enhanced tumor regressions, compared with treatments with either agent alone. While anti-PD-1 did not reduce the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells present in tumor-bearing mice, we found that it increased expression of IFN-γ and CXCL10 at the tumor site. Bone marrow-transplant experiments using IFN-γR-/- mice implicated IFN-γ as a crucial nexus for controlling PD-1-mediated tumor infiltration by T cells. Taken together, our results imply that blocking the PD-1 pathway can increase IFN-γ at the tumor site, thereby increasing chemokine-dependent trafficking of immune cells into malignant disease sites.

Oncogenic BRAF(V600E) promotes stromal cell-mediated immunosuppression via induction of interleukin-1 in melanoma.

PURPOSE: In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF). EXPERIMENTAL DESIGN: Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. RESULTS: Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1ß in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/ß significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs. CONCLUSIONS: This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.