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Lack of response and acquired resistance continue to be limitations of targeted and immune-based therapies. Pyroptosis is an inflammatory form of cell death characterized by the release of inflammatory damage-associated molecular patterns (DAMP) and cytokines via gasdermin (GSDM) protein pores in the plasma membrane. Induction of pyroptosis has implications for treatment strategies in both therapy-responsive, as well as resistance forms of melanoma. We show that the caspase-3 activator, raptinal, induces pyroptosis in both human and mouse melanoma cell line models and delays tumor growth in vivo. Release of DAMPs and inflammatory cytokines was dependent on caspase activity and GSDME expression. Furthermore, raptinal stimulated pyroptosis in melanoma models that have acquired resistance to BRAF and MEK inhibitor therapy. These findings add support to efforts to induce pyroptosis in both the treatment-naïve and resistant settings. Implications: Raptinal can rapidly induce pyroptosis in naïve and BRAFi plus MEKi-resistant melanoma, which may be beneficial for patients who have developed acquired resistance to targeted therapies.

Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDA-approved to treat BRAFV600E/K-mutant melanoma. Efficacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immune-mediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-deficient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infiltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi–resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infiltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi–induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma.Significance:Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma.See related commentary by Smalley, p. 176.This article is highlighted in the In This Issue feature, p. 161

Supplementary Figure from Raptinal Induces Gasdermin E–Dependent Pyroptosis in Naïve and Therapy-Resistant Melanoma

Supplementary Figures and Legends.

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Melanomas frequently harbor activating NRAS mutations. However, limited advance has been made in developing targeted therapy options for patients with NRAS mutant melanoma. MEK inhibitors (MEKi) show modest efficacy in the clinic and their actions need to be optimized. In this study, we performed a genome-wide CRISPR-Cas9–based screen and demonstrated that loss of phosphoinositide-dependent kinase-1 (PDPK1) enhances the efficacy of MEKi. The synergistic effects of PDPK1 loss and MEKi was validated in NRAS mutant melanoma cell lines using pharmacologic and molecular approaches. Combined PDPK1 inhibitors (PDPK1i) with MEKi suppressed NRAS mutant xenograft growth and induced gasdermin E–associated pyroptosis. In an immune-competent allograft model, PDPK1i+MEKi increased the ratio of intratumoral CD8+ T cells, delayed tumor growth, and prolonged survival; the combination treatment was less effective against tumors in immune-deficient mice. These data suggest PDPK1i+MEKi as an efficient immunostimulatory strategy against NRAS mutant melanoma. Significance: Targeting PDPK1 stimulates antitumor immunity and sensitizes NRAS mutant melanoma to MEK inhibition, providing rationale for the clinical development of a combinatorial approach for treating patients with melanoma.

Psychological pharmacology is a brand-new discipline that studies the interaction between psychological state and drugs，explores general rules of the effects of mental state on drug activity，and promotes rational use of drugs in clinical practice.Evidence showed that depression could affect the levels of endogenous substances，drug metabolizing enzymes and drug transport protein，thus influencing drug metabolism and efficacy.In clinical practice，medication recognition might affect drug choice and medication compliance of patients.The application of placebo might achieve positive treatment outcomes in certain patients.These research results indicate that attention should be paid to the psychological state of patients in drug treatment.In accordance with changes of physiological and psychological changes of patients，therapeutic regimens should be duly adjusted.Rational drug use education and psychological support should be provided to patients，so that patients’ psychological burden could be relieved and therapeutic efficacy improved.

SummaryApproaches systematically characterizing interactions via transcriptomic data usually follow two systems: (1) co-expression network analyses focusing on correlations between genes; (2) linear regressions (usually regularized) to select multiple genes jointly. Both suffer from the problem of stability: a slight change of parameterization or dataset could lead to dramatic alternations of outcomes. Here, we propose Stabilized Core gene and Pathway Election, or SCOPE, a tool integrating bootstrapped LASSO and co-expression analysis, leading to robust outcomes insensitive to variations in data. By applying SCOPE to six cancer expression datasets (BRCA, COAD, KIRC, LUAD, PRAD and THCA) in The Cancer Genome Atlas, we identified core genes capturing interaction effects in crucial pan-cancer pathways related to genome instability and DNA damage response. Moreover, we highlighted the pivotal role of CD63 as an oncogenic driver and a potential therapeutic target in kidney cancer. SCOPE enables stabilized investigations towards complex interactions using transcriptome data.Availabilityhttps://github.com/QingrunZhangLab/SCOPE

Despite the clinical success of targeted inhibitors, tumor responses to these agents are transient, and drug-tolerant residual cells seed resistance. Understanding the role of tumor-intrinsic mechanisms and effects of the tumor microenvironment in mediating drug tolerance will guide and optimize targeted therapies. Given similarities between drug tolerance and cellular dormancy, we studied the role of nuclear receptor subfamily 2 group F member 1 (NR2F1) in response to targeted therapy. We used BRAF-mutant cutaneous melanoma models treated with BRAF and MEK inhibitors (BRAFi + MEKi) since patients treated with this combination typically develop resistance. The aged tumor microenvironment has been shown to increase therapy resistance, and we find that melanoma cells in aged mice express higher levels of NR2F1 than when the same cells are injected into young animals. Transcriptomic analysis of melanoma patient samples treated with BRAFi + MEKi showed increased expression of NR2F1 post-treatment. Similarly, NR2F1 was highly expressed in minimal residual disease collected on BRAFi + MEKi treatment in patient- and xenograft-derived tumors. High expression of NR2F1 promotes tumor survival and invasion in the presence of BRAFi + MEKi in vitro leading to tolerance to BRAFi + MEKi efficacy in vivo. Depletion of NR2F1 in YUMM1.7 allografts grown in aged mice improved response to the combination therapy. Altogether, our findings suggest that NR2F1 promotes drug tolerance leading to minimal residual disease in melanoma and that NR2F1-high cells may be targeted with CDK4/6 inhibitors to improve targeted therapy outcomes in melanoma patients. Citation Format: Manoela Tiago, Timothy J. Purwin, Mitchell E. Fane, Yash Chhabra, Jessica L. F. Teh, Rama Kadamb, Weijia Cai, Inna Chervoneva, Sheera Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanieva, Ashani T. Weeraratna, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin. The aged tumor microenvironment influences tolerance to targeted therapy via NR2F1 overexpression in BRAF-mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A005.

Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDA-approved to treat BRAFV600E/K-mutant melanoma. Efficacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immune-mediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-deficient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infiltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi–resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infiltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi–induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma. Significance: Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma. See related commentary by Smalley, p. 176. This article is highlighted in the In This Issue feature, p. 161

p53 acetylation is indispensable for its transcriptional activity and tumor suppressive function. However, the identity of reader protein(s) for p53 acetylation remains elusive. PBRM1, the second most highly mutated tumor suppressor gene in kidney cancer, encodes PBRM1. Here, we identify PBRM1 as a reader for p53 acetylation on lysine 382 (K382Ac) through its bromodomain 4 (BD4). Notably, mutations on key residues of BD4 disrupt recognition of p53 K382Ac. The mutation in BD4 also reduces p53 binding to promoters of target genes such as CDKN1A (p21). Consequently, the PBRM1 BD4 mutant fails to fully support p53 transcriptional activity and is defective as a tumor suppressor. We also find that expressions of PBRM1 and p21 correlate with each other in human kidney cancer samples. Our findings uncover a tumor suppressive mechanism of PBRM1 in kidney cancer and provide a mechanistic insight into the crosstalk between p53 and SWI/SNF complexes., Acetylation of p53 is critical for its transcriptional activity and its tumour suppressive function. Here, the authors show that PBRM1 is a reader protein for p53′s C-terminal domain acetylation on lysine 382 through its bromodomain 4 and that mutations in this domain leads to compromised tumour suppressive function and renal tumour growth.

The calibration of lenslet-based light field camera is the key issue to many applications, especially the three-dimensional shape measurement. In this paper, a two-step physically based calibration method is proposed, where every parameter is described with its own physical meaning. The parameters about the main lens of the light field camera are calibrated with the central sub-aperture image, and the parameters about the micro-lens array are calibrated with the epipolar images, subsequently. To improve the calibration result, the distortion correction of the main lens and a nonlinear optimization method are applied also. The calibration method is validated with a commercially available light field camera and compared to Dansereau's calibration method. Typical RMS reprojection errors are 0.0017, 0.0032 mm for 30.0, 35.1 mm calibration grids.

In our previous study, human fibroblast growth factor 1 was successfully fused with oleosomes, energy-storing organelles of seeds, which are considered to be excellent “expression carriers” for substances with a convenient purification process. The present work aimed to explore the beneficial effects of oleosomes fused with human fibroblast growth factor 1 (OLAF) on wound healing. The data showed marked improvements in terms of the angiogenesis, vascular integrity, collagen and inflammation on the wound sites of rats with a full-thickness skin defect. Moreover, the positive role of OLAF in promoting angiogenesis and its possible pathways were clarified in vivo and in vitro. The results showed that the number, length and branches of the blood vessels of the chick embryo chorioallantoic membrane were markedly increased after OLAF treatment. Meanwhile, the in vitro results also revealed that 100 ng/mL OLAF exhibited a promoting effect on the proliferation, migration and tube formation of human umbilical vein endothelial cells. In addition, the potential of OLAF to improve wound angiogenesis was demonstrated to be associated with an up-regulated PI3K/Akt pathway by transcriptome sequencing analysis and the introduction of a PI3K/Akt pathway inhibitor (LY294002). These findings suggest that OLAF has many prospects in the development of drugs for wound healing.

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo. These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance. Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells.

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Three-dimensional light field imaging in laparoscopic surgery is an emerging technology, which has the potential to enable three-dimensional imaging. Calibration of the three-dimensional light field endoscope (3D LFE) is essential but challenging as the disparity is much smaller than that of the conventional light field camera. The geometrical model for 3D LFE is established, and a calibration method based on virtual objective lens and virtual feature points is proposed. First, the virtual objective lens is introduced and the parameters about it are calibrated using corner features in center subaperture images. Second, two types of virtual feature points are proposed to calibrate the parameters about the microlens array, one is on the black-and-white board line and the other is selectively determined but can be anywhere on the checkerboard. Moreover, the relationship between the virtual feature points mapping in the microlens image and the virtual feature points mapping in the central subaperture image is deduced to overcome tiny light field disparity. Experimental results verify the performance of our calibration method.

Four graphene oxide samples with different oxygenated groups are prepared and characterized, and then employed as the additives in the negative active materials to investigate the effect of different oxygenated groups of graphene oxide on the H2 evolution performance of negative plates and the high-rate partial-state-of-charge cycle life of simulated lead-acid batteries. The results indicate that the H2 evolution and the reduction of PbSO4 processes are largely accelerated by the graphene oxide additives. An increase in C O groups (C OH and C O C) of graphene oxide can largely promote the H2 evolution process. The addition of the graphene oxide additives largely increases the surface area and total pore volume of the negative plates, meanwhile obviously increases the hydrophilicity of negative active materials to facilitate the diffusion of acid into the inner of the plate. The high-rate partial-state-of-charge cycle life of the simulated test cells containing the graphene oxide samples is prolonged significantly, and especially those containing graphene oxide with less C O groups and more carbonyl and carboxyl groups have the longest cycle life, which seems to be more appropriate as the additive of negative plates.

Melanomas frequently harbor activating NRAS mutations; however, there has been little advance in targeted therapy options for NRAS mutant melanoma patients. MEK inhibitors (MEKi) showed modest efficacy in clinic, which is insufficient to be approved by FDA. In this study, we performed a genome-wide CRISPR/Cas9-based screening, identified PDPK1 (Phosphoinositide-dependent kinase-1) as a therapeutic target to enhancing the efficacy of MEKi, and validated it in various NRAS mutant melanoma cell lines via pharmacological and genetic approaches. Combined inhibition of PDPK1 and MEK (PDPK1i+MEKi) profoundly inhibited NRAS mutant tumor growth in a xenograft model. Notably, the combinatorial treatment induced pyroptosis, and increased ratio of intratumoral CD8+ T cells, delayed tumor growth and prolonged survival in an immune competent allograft model whereas it showed a significantly weaker potency in an isogenic immune deficient model. These data suggest PDPK1i + MEKi is an efficient strategy against NRAS mutant melanoma in an immune-response-dependent manner. Significance: NRAS is still an 'undruggable' target in melanoma. Our discovery rationalizes the clinical development of PDPK1i plus MEKi in NRAS mutant melanoma patients and suggests further synergy of targeted therapy and immunotherapy. Citation Format: Weijia Cai, Mai Nguyen, nicole wilski, timothy purwin, Manoela TiagodosSantos, Andrew Aplin. Genetic screen identifies PDPK1 as a synergistic target to enhancing the efficacy of MEK1/2 inhibitors in NRAS mutant melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P180.

In monocular structured light system, the iterative reconstruction method makes the measurement time-consuming, and the measurement accuracy is often hindered by the fact that the first-order radial distortion is included only. Compared with the conventional projector model, a new projector model, including both radial and tangential distortion, is proposed in this paper, which is described with the pinhole model according to the light direction. Furthermore, the iterative method is replaced by solving a quartic polynomial problem directly based on the proposed projector model. Experimental results show that the measurement accuracy and the efficiency are improved obviously. The standard deviation of the proposed method is 0.037mm, which is about a third of 0.113mm of the iterative method. The time consumed by the proposed method is 3.3% of that by the iterative method when one hundred thousand points are reconstructed.

In this paper, a three-dimensional (3D) shape measurement method based on structured light field imaging is proposed. Generally, light field imaging is challenging to accomplish the 3D shape measurement accurately, as the slope estimation method based on radiance consistency is inaccurate. Taking into account the special modulation of structured light field, the phase information is derived with Fourier transform profilometry, which is utilized to substitute the phase consistency for the radiance consistency in epipolar image (EPI) at first. Therefore, the 3D coordinates are derived after light field calibration, but the results are coarse due to slope estimation error and need to be corrected. Furthermore, the 3D coordinates refinement is performed based on relationship between the structured light field image and DMD image of the projector, which allows to improve the performance of the 3D shape measurement. The necessary light field camera calibration is described to generalize its application. Subsequently, the effectiveness of the proposed method is demonstrated with a sculpture and compared to the results of a conventional PMP system.

Light field camera calibration is much more complicated by the fact that a single point in the 3D scene appears many times in the image plane. Compared to the previous geometrical models of light field camera, which describe the relationship between 3D point in the scene and 4D light field, we proposed an epipolar-space (EPS) based geometrical model in this paper, which determines the relationship between 3D point in the scene and 3-parameter vector in the EPS. Moreover, a close-form solution for the 3D shape measurement based on the geometrical model is accomplished. Our calibration method includes an initial linear solution and nonlinear optimization with the Levenberg-Marquardt algorithm. The light field model is validated with the commercially available light field camera Lytro iIIum, and the performance of 3D shape measurement is verified by both real scene data and the data set on the internet.

Intratumoral heterogeneity and cellular plasticity enable tumors to alter phenotypes and adapt to foreign microenvironments and resist targeted inhibitors. While the ability to switch between phenotypic states has been broadly characterized, the key mechanisms that underlie tumor plasticity remain poorly understood. We studied the neural crest lineage transcription factor, SOX10, in the context of cutaneous melanoma and resistance to targeted therapies. SOX10 is heterogeneously expressed in melanoma samples. Using bio-informatics as well as in vivo and 3D in vitro melanoma models, SOX10 loss was sufficient to induce an invasive but slow proliferating phenotype in vitro and in vivo that was associated with expression of a mesenchymal gene set. Interestingly, while SOX10 knockout initially induced a targeted inhibitor tolerant state, longer exposure of co-mixed populations of SOX10 proficient and SOX10 deficient to targeted therapy drives the clonal selection of SOX10 knockout cells. Furthermore, cell lines generated from xenograft tumors that have acquired resistance to either vemurafenib, paradox-breaking BRAFi or the combination of BRAFi + MEKi showed dramatically reduced SOX10 expression compared to their parental counterparts. Altogether these data suggest that acquired resistant clones may arise from drug tolerant persister cells. As a strategy to selectively target this invasive, drug-tolerant SOX10-deficient sub-population, we screened a drug compound library and identified a class of cIAP1/2 inhibitors to be synthetically lethal for SOX10-deficient cells. Our preliminary data suggest that birinapant can delay or prevent resistance to BRAFi/MEKi in vivo. Together, these data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma and enables tumor adaptation to altered microenvironments and drug treatments which could be targeted using cIAP1/2 inhibitors. Citation Format: Claudia Capparelli, Timothy J. Purwin, Manoela Tiago, Nicole Wilski, Danielle Pomante, McKenna Glasheen, Sheera Rosenbaum, Mai Q. Nguyen, Weijia Cai, Richard Zheng, Gaurav Kumar, Inna Chervoneva, Ayako Shimada, Adam E. Snook, Paolo Fortina, Xiaowei Xu, Kim Hookim, Edna Cukierman, Michael A. Davies, Meenhard Herlyn, Andrew E. Aplin. Targeting SOX10-deficient cells to reduce resistance to targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 40.

Despite the clinical success of targeted therapy and checkpoint inhibitors in melanoma, therapeutic responses are transient, followed by relapse that may be driven by a small subpopulation of residual or drug-tolerant cells. Understanding residual disease and metastasis mechanisms can provide clues both for developing improved versions of a drug and for guiding the selection of appropriate drug combinations for melanoma therapy. Here, we found that the well-known marker of tumor dormancy, Nuclear Receptor Subfamily 2 Group F Member 1 (NR2F1), was overexpressed in minimal disease residual cells following CDK4/6 and MEK inhibitors (CDK4/6i+MEKi) treatment in vivo. Furthermore, melanoma cells overexpressing NR2F1 were less sensitive to (CDK4/6i+MEKi) or BRAF and MEK inhibitors (BRAFi+MEKi) treatment in vitro and in vivo models, inhibiting apoptosis. Surprisingly, we did not find any evidence of decreased cell growth in our model. Using a three-dimensional tumor spheroid assay in vitro, we found the NR2F1 expression enhanced melanoma invasion following CDK4/6i+MEKi or BRAFi+MEKi treatments. The use of published RNA Seq data sets that were gathered from the GEO database and Single Cell Seq data sets from PDX melanoma samples showed that high expression of NR2F1 is enriched in the undifferentiated cell state and invasive cells, respectively. Furthermore, BRAF mutant patient sample with an acquired mutation in NRAS Q61R following BRAFi+MEKi+CDKi presented a high expression of NR2F1. Altogether, these findings suggest that NR2F1 may play a role in residual disease persistence besides known features of tumor dormancy, especially important in determining responses to dramatic changes in the environment, such as changes induced by anti-cancer therapy. Citation Format: Manoela Tiago, Jessica L. Teh, Timothy J. Purwin, Weijia Cai, Connor Hollingworth, Melisa Lopez-Anton, Julio A. Aguirre-Ghiso, Andrew E. Aplin. NR2F1 underlies persistence of residual disease in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2986.