A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

IL-7R licenses a population of epigenetically poised memory CD8+ T cells with superior antitumor efficacy that are critical for melanoma memory.

Recurrence of advanced melanoma after therapy is a major risk factor for reduced survival, and treatment options are limited. Antitumor immune memory plays a critical role in preventing melanoma recurrence and memory T cells could be a potent cell-based therapy, but the identity, and functional properties of the required immune cells are incompletely understood. Here, we show that an IL-7Rhi tumor-specific CD8+ population is critical for antitumor memory and can be epigenetically augmented to drive powerful antitumor immune responses. Using a model of functional antimelanoma memory, we found that high IL-7R expression selectively marks a CD8+ population in lymphoid organs that plays critical roles in maintaining tumor remission after immunotherapy or surgical resection. This population has intrinsic cytotoxic activity, lacks markers of exhaustion and has superior antitumor efficacy. IL-7Rhi cells have a functionally poised epigenetic landscape regulated by DNA methylation, which can be augmented by hypomethylating agents to confer improved survival and complete melanoma clearance in naive mice. Importantly, greater than 95% of tumor-specific T cells in draining lymph nodes after therapy express high levels of IL-7R. This overlap between IL-7Rhi and antigen-specific T cells allows for enrichment of a potent functional CD8+ population without determining antigen-specificity, which we demonstrate in a melanoma model without a known antigen. We identify that IL-7R expression in human melanoma is an independent prognostic factor of improved survival. These findings advance our basic understanding of antitumor memory and suggest a cell-based therapy using high IL-7R expression to enrich for a lymph node population with superior antitumor activity that can be augmented by hypomethylating agents.

Heterogeneous Expression and Subcellular Localization of Pyruvate Dehydrogenase Complex in Prostate Cancer.

Background: Pyruvate dehydrogenase (PDH) complex converts pyruvate into acetyl-CoA by pyruvate decarboxylation, which drives energy metabolism during cell growth, including prostate cancer (PCa) cell growth. The major catalytic subunit of PDH, PDHA1, is regulated by phosphorylation/dephosphorylation by pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs). There are four kinases, PDK1, PDK2, PDK3 and PDK4, which can phosphorylate and inactivate PDH; and two phosphatases, PDP1 and PDP2, that dephosphorylate and activate PDH. Methods: We have analyzed by immunohistochemistry the expression and clinicopathological correlations of PDHA1, PDP1, PDP2, PDK1, PDK2, PDK3, and PDK4, as well as of androgen receptor (AR), in a retrospective PCa cohort of patients. A total of 120 PCa samples of representative tumor areas from all patients were included in tissue microarray (TMA) blocks for analysis. In addition, we studied the subcellular localization of PDK2 and PDK3, and the effects of the PDK inhibitor dichloroacetate (DCA) in the growth, proliferation, and mitochondrial respiration of PCa cells. Results: We found heterogeneous expression of the PDH complex components in PCa tumors. PDHA1, PDP1, PDK1, PDK2, and PDK4 expression correlated positively with AR expression. A significant correlation of PDK2 immunostaining with biochemical recurrence and disease-free survival was revealed. In PCa tissue specimens, PDK2 displayed cytoplasmic and nuclear immunostaining, whereas PDK1, PDK3 and PDK4 showed mostly cytoplasmic staining. In cells, ectopically expressed PDK2 and PDK3 were mainly localized in mitochondria compartments. An increase in maximal mitochondrial respiration was observed in PCa cells upon PDK inhibition by DCA, in parallel with less proliferative capacity. Conclusion: Our findings support the notion that expression of specific PDH complex components is related with AR signaling in PCa tumors. Furthermore, PDK2 expression associated with poor PCa prognosis. This highlights a potential for PDH complex components as targets for intervention in PCa.

Progress and challenges for the machine learning-based design of fit-for-purpose monoclonal antibodies.

Although the therapeutic efficacy and commercial success of monoclonal antibodies (mAbs) are tremendous, the design and discovery of new candidates remain a time and cost-intensive endeavor. In this regard, progress in the generation of data describing antigen binding and developability, computational methodology, and artificial intelligence may pave the way for a new era of in silico on-demand immunotherapeutics design and discovery. Here, we argue that the main necessary machine learning (ML) components for an in silico mAb sequence generator are: understanding of the rules of mAb-antigen binding, capacity to modularly combine mAb design parameters, and algorithms for unconstrained parameter-driven in silico mAb sequence synthesis. We review the current progress toward the realization of these necessary components and discuss the challenges that must be overcome to allow the on-demand ML-based discovery and design of fit-for-purpose mAb therapeutic candidates.

Antibody variable sequences have a pronounced effect on cellular transport and plasma half-life.

Monoclonal IgG antibodies are the fastest growing class of biologics, but large differences exist in their plasma half-life in humans. Thus, to design IgG antibodies with favorable pharmacokinetics, it is crucial to identify the determinants of such differences. Here, we demonstrate that the variable region sequences of IgG antibodies greatly affect cellular uptake and subsequent recycling and rescue from intracellular degradation by endothelial cells. When the variable sequences are masked by the cognate antigen, it influences both their transport behavior and binding to the neonatal Fc receptor (FcRn), a key regulator of IgG plasma half-life. Furthermore, we show how charge patch differences in the variable domains modulate both binding and transport properties and that a short plasma half-life, due to unfavorable charge patches, may partly be overcome by Fc-engineering for improved FcRn binding.

Soluble AXL as a marker of disease progression and survival in melanoma.

Receptor tyrosine kinase AXL is a one-pass transmembrane protein upregulated in cancers and associated with lower survival and therapy resistance. AXL can be cleaved by the A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, yielding a soluble version of the protein. Elevated soluble AXL (sAXL) has been reported to be associated with disease progression in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases. In the present work, we analyzed sAXL levels in blood from melanoma patients and showed that sAXL increases with disease progression. Additionally, increased sAXL levels were found correlated with shorter two-year survival in stage IV patients treated with ipilimumab. Furthermore, we showed that sAXL levels were related to the percentage of cells expressing AXL in resected melanoma lymph node metastases. This finding was verified in vitro, where sAXL levels in the cell media corresponded to AXL expression in the cells. AXL inhibition using the small-molecular inhibitor BGB324 reduced sAXL levels, while the cellular expression was elevated through increased protein stability. Our findings signify that quantification of sAXL blood levels is a simple and easily assessable method to determine cellular AXL levels and should be further evaluated for its use as a biomarker of disease progression and treatment response.

B7-H3 Immune Checkpoint Protein in Human Cancer.

B7-H3 belongs to the B7 family of immune checkpoint proteins, which are important regulators of the adaptive immune response and emerging key players in human cancer. B7-H3 is a transmembrane protein expressed on the surface of tumor cells, antigen presenting cells, natural killer cells, tumor endothelial cells, but can also be present in intra- and extracellular vesicles. Additionally, B7-H3 may be present as a circulating soluble isoform in serum and other body fluids. B7-H3 is overexpressed in a variety of tumor types, in correlation with poor prognosis. B7-H3 is a promising new immunotherapy target for anti-cancer immune response, as well as a potential biomarker. Besides its immunoregulatory role, B7-H3 has intrinsic pro-tumorigenic activities related to enhanced cell proliferation, migration, invasion, angiogenesis, metastatic capacity and anti-cancer drug resistance. B7-H3 has also been found to regulate key metabolic enzymes, promoting the high glycolytic capacity of cancer cells. B7-H3 receptors are still not identified, and little is known about the molecular mechanisms underlying B7-H3 functions. Here, we review the current knowledge on the involvement of B7-H3 in human cancer.

Targeting AXL and the DNA Damage Response Pathway as a Novel Therapeutic Strategy in Melanoma.

Receptor tyrosine kinase AXL is found upregulated in various types of cancer, including melanoma, and correlates with an aggressive cancer phenotype, inducing cell proliferation and epithelial-to-mesenchymal transition. In addition, AXL has recently been linked to chemotherapy resistance, and inhibition of AXL is found to increase DNA damage and reduce expression of DNA repair proteins. In light of this, we aimed to investigate whether targeting AXL together with DNA damage response proteins would be therapeutically beneficial. Using melanoma cell lines, we observed that combined reduction of AXL and CHK1/CHK2 signaling decreased proliferation, deregulated cell-cycle progression, increased apoptosis, and reduced expression of DNA damage response proteins. Enhanced therapeutic effect of combined treatment, as compared with mono-treatment, was further observed in a patient-derived xenograft model and, of particular interest, when applying a three-dimensional ex vivo spheroid drug sensitivity assay on tumor cells harvested directly from 27 patients with melanoma lymph node metastases. Together, these results indicate that targeting AXL together with the DNA damage response pathway could be a promising treatment strategy in melanoma, and that further investigations in patient groups lacking treatment alternatives should be pursued.

A Three-dimensional Ex Vivo Viability Assay Reveals a Strong Correlation Between Response to Targeted Inhibitors and Mutation Status in Melanoma Lymph Node Metastases.

Although clinical management of melanoma has changed considerably in recent years, intrinsic treatment resistance remains a severe problem and strategies to design personal treatment regimens are highly warranted. We have applied a three-dimensional (3D) ex vivo drug efficacy assay, exposing disaggregated cells from 38 freshly harvested melanoma lymph node metastases and 21 patient derived xenografts (PDXs) to clinical relevant drugs for 7 days, and examined its potential to evaluate therapy response. A strong association between Vemurafenib response and BRAF mutation status was achieved (P < .0001), while enhanced viability was seen in some NRAS mutated tumors. BRAF and NRAS mutated tumors responded comparably to the MEK inhibitor Cobimetinib. Based on the ex vivo results, two tumors diagnosed as BRAF wild-type by routine pathology examinations had to be re-evaluated; one was subsequently found to have a complex V600E mutation, the other a double BRAF mutation (V600E/K601 N). No BRAF inhibitor resistance mechanisms were identified, but PIK3CA and NF1 mutations were identified in two highly responsive tumors. Concordance between ex vivo drug responses using tissue from PDXs and corresponding patient tumors demonstrate that PDX models represent an indefinite source of tumor material that may allow ex vivo evaluation of numerous drugs and combinations, as well as studies of underlying molecular mechanisms. In conclusion, we have established a rapid and low cost ex vivo drug efficacy assay applicable on tumor tissue from patient biopsies. The 3D/spheroid format, limiting the influence from normal adjacent cells and allowing assessment of drug sensitivity to numerous drugs in one week, confirms its potential as a supplement to guide clinical decision, in particular in identifying non-responding patients.

p38 MAPK activation through B7-H3-mediated DUSP10 repression promotes chemoresistance.

Immunoregulatory protein B7-H3 is involved in the oncogenic and metastatic potential of cancer cells, as well as in drug resistance. Resistance to conventional chemotherapy is an important aspect of melanoma treatment, and a better understanding of how B7-H3 enhances drug resistance may lead to the development of more effective therapies. We investigated the in vitro and in vivo sensitivity of chemotherapeutic agents dacarbazine (DTIC) and cisplatin in sensitive and drug resistant melanoma cells with knockdown expression of B7-H3. We found that knockdown of B7-H3 increased in vitro and in vivo sensitivity of melanoma cells to the chemotherapeutic agents dacarbazine (DTIC) and cisplatin, in parallel with a decrease in p38 MAPK phosphorylation. Importantly, in B7-H3 knockdown cells we observed an increase in the expression of dual-specific MAP kinase phosphatase (MKP) DUSP10, a MKP known to dephosphorylate and inactivate p38 MAPK. DUSP10 knockdown by siRNA resulted in a reversion of the increased DTIC-sensitivity seen in B7-H3 knockdown cells. Our findings highlight the potential therapeutic benefit of combining chemotherapy with B7-H3 inhibition, and indicate that B7-H3 mediated chemoresistance in melanoma cells is driven through a mechanism involving DUSP10-mediated inactivation of p38 MAPK.

Soluble AXL is ubiquitously present in malignant serous effusions.

OBJECTIVE: The objective of this study was to analyze the expression level and clinical role of soluble AXL (sAXL) in cancers affecting the serosal surfaces, with focus on ovarian carcinoma. METHODS: sAXL protein expression by ELISA was analyzed in 572 effusion supernatants, including 424 peritoneal, 147 pleural and 1 pericardial specimens. RESULTS: sAXL was overexpressed in peritoneal effusions compared to pleural and pericardial specimens (p < 0.001). sAXL levels were additionally significantly higher in effusions from patients with ovarian carcinoma, malignant mesothelioma and breast carcinoma compared to specimens from patients with other cancers (predominantly carcinomas of lung, gastrointestinal or uterine corpus/cervix origin) or benign reactive effusions (p < 0.001). sAXL was further overexpressed in high-grade serous carcinoma (HGSC; n = 373) compared to low-grade serous carcinoma (LGSC; n = 32; p = 0.036). In HGSC, sAXL levels were significantly lower in post-chemotherapy effusions compared to primary diagnosis pre-chemotherapy specimens (p = 0.002). sAXL levels in HGSC were unrelated to chemoresponse at diagnosis, progression-free survival or overall survival. Levels were similarly unrelated to survival in LGSC and breast carcinoma. CONCLUSIONS: sAXL is widely expressed in malignant effusions, particularly in ovarian and breast carcinoma and in malignant mesothelioma. sAXL is overexpressed in HGSC compared to LGSC and its levels are lower following exposure to chemotherapy. However, sAXL levels are not informative of chemoresponse or survival.

B7-H3 in Cancer - Beyond Immune Regulation.

B7-H3, a member of the B7 family of immunoregulatory proteins, is overexpressed in cancer and promotes tumor growth, metastasis, and drug resistance. We discuss here the involvement of B7-H3 in cancer that goes beyond its immune regulatory function, and discuss the potential of B7-H3 as a biomarker and therapeutic target.

Immunoregulatory protein B7-H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells.

B7-H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7-H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small-molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK-162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API-2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7-H3 monoclonal antibody, while the opposite was seen in B7-H3-overexpressing cells. Further, combining B7-H3 inhibition with small-molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAFV600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7-H3 may be a novel alternative to improve current therapy of metastatic melanoma.