Single-cell expression profiles of melanoma Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science, this issue p. 189 Melanoma cells show transcriptional heterogeneity. To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

PurposeTherapeutic selectivity is one of the most important considerations in cancer chemotherapy. The design of therapeutic strategies to preferentially kill malignant cells while minimizing harmful effects to normal cells depends on our understanding of the biological differences between cancer and normal cells. We have previously demonstrated that certain agents generating reactive oxygen species (ROS) such as 2-methoxyestradiol (2-ME) preferentially kill human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The purpose of the current study was to investigate the biochemical basis for such selective anticancer activity.MethodsFlow cytometric analyses were utilized to measure intracellular O2− levels and apoptosis. MTT assays were used as indicators of cellular viability. Western blot analysis was used to measure the expression of antioxidant enzymes in cancer and normal cells.ResultsMalignant cells in general are more active than normal cells in the production of O2−, are under intrinsic oxidative stress, and thus are more vulnerable to damage by ROS-generating agents. The intrinsic oxidative stress in cancer cells was associated with the upregulation of SOD and catalase protein expression, likely as a mechanism to tolerate increased ROS stress. The increase in SOD and catalase expression was observed both in primary human leukemia cells and in primary ovarian cancer cells. Both malignant cell types were more sensitive to 2-ME than their normal counterparts, as demonstrated by the significant accumulation of O2− and subsequent apoptosis. The administration of ROS scavengers in combination with 2-ME prevented the accumulation of O2− and abrogated apoptosis induction.ConclusionsO2− is an important mediator of 2-ME-induced apoptosis. The increased oxidative stress in cancer cells forces these cells to rely more on antioxidant enzymes such as SOD for O2− elimination, thus making the malignant cells more vulnerable to SOD inhibition than normal cells.

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

Advances in HER2-targeted therapies have improved the survival of patients with HER2-positive breast cancer. The standard-of-care treatment for localized disease has been chemotherapy and 1 year of adjuvant HER2-targeted therapy, typically with the anti-HER2 antibody trastuzumab. Despite the effectiveness of this treatment, disease relapse occurs in a subset of patients; thus, focus has been placed on escalating treatment by either combining different HER2-targeted agents or extending the duration of HER2-targeted therapy. Indeed, dual HER2-targeted therapies and extended-duration anti-HER2 therapy, as well as adjuvant therapy with the anti-HER2 antibody–drug conjugate T-DM1, have all been approved for clinical use. Emerging evidence suggests, however, that some patients do not derive sufficient benefit from these additional therapies to offset the associated toxicities and/or costs. Similarly, the universal use of chemotherapy might not benefit all patients, and treatment de-escalation through omission of chemotherapy has shown promise in clinical trials and is currently being explored further. The future of precision medicine should therefore involve tailoring of therapy based on the genetics and biology of each tumour and the clinical characteristics of each patient. Predictive biomarkers that enable the identification of patients who will benefit from either escalated or de-escalated treatment will be crucial to this approach. In this Review, we summarize the available HER2-targeted agents and associated mechanisms of resistance, and describe the current therapeutic landscape of early stage HER2-positive breast cancer, focusing on strategies for treatment escalation or de-escalation. HER2-targeted therapy has greatly improved the outcomes of patients with HER2-positive breast cancer, with a range of agents now approved or in late-stage clinical development. In the era of precision medicine, efforts are being made to further improve patient outcomes by personalizing HER2-targeted treatment regimens, primarily though escalation or de-escalation of therapy according to the disease biology. In this Review, the authors provide an overview of the current landscape of HER2-targeted therapy and discuss the evidence supporting such tailored therapeutic strategies.

Towards personalized treatment for early stage HER2-positive breast cancer

The epidermal growth factor receptor (EGFR) family contains four transmembrane tyrosine kinases (EGFR1/ErbB1, Her2/ErbB2, Her3/ErbB3 and Her4/ErbB4) and 13 secreted polypeptide ligands. EGFRs are overexpressed in many solid tumors, including breast, pancreas, head-and-neck, prostate, ovarian, renal, colon, and non-small-cell lung cancer. Such overexpression produces strong stimulation of downstream signaling pathways, which induce cell growth, cell differentiation, cell cycle progression, angiogenesis, cell motility and blocking of apoptosis.The high expression and/or functional activation of EGFRs correlates with the pathogenesis and progression of several cancers, which make them attractive targets for both diagnosis and therapy. Several approaches have been developed to target these receptors and/or the EGFR modulated effects in cancer cells. Most approaches include the development of anti-EGFRs antibodies and/or small-molecule EGFR inhibitors. This review presents the state-of-the-art and future prospects of targeting EGFRs to treat breast cancer.

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Molecular Targeting Therapy against EGFR Family in Breast Cancer: Progress and Future Potentials

Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage–derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.

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Phage Display Derived Monoclonal Antibodies: From Bench to Bedside.

ERBB family members including epidermal growth factor receptor (EGFR) also known as HER1, ERBB2/HER2/Neu, ERBB3/HER3 and ERBB4/HER4 are aberrantly activated in multiple cancers and hence serve as drug targets and biomarkers in modern precision therapy. The therapeutic potential of HER3 has long been underappreciated, due to impaired kinase activity and relatively low expression in tumors. However, HER3 has received attention in recent years as it is a crucial heterodimeric partner for other EGFR family members and has the potential to regulate EGFR/HER2-mediated resistance. Upregulation of HER3 is associated with several malignancies where it fosters tumor progression via interaction with different receptor tyrosine kinases (RTKs). Studies also implicate HER3 contributing significantly to treatment failure, mostly through the activation of PI3K/AKT, MAPK/ERK and JAK/STAT pathways. Moreover, activating mutations in HER3 have highlighted the role of HER3 as a direct therapeutic target. Therapeutic targeting of HER3 includes abrogating its dimerization partners' kinase activity using small molecule inhibitors (lapatinib, erlotinib, gefitinib, afatinib, neratinib) or direct targeting of its extracellular domain. In this review, we focus on HER3-mediated signaling, its role in drug resistance and discuss the latest advances to overcome resistance by targeting HER3 using mono- and bispecific antibodies and small molecule inhibitors.

https://www.oncologyreviews.org/site/article/download/355/334

HER3 signaling and targeted therapy in cancer.

Melanoma is the most lethal form of skin cancer. Melanoma is usually curable with surgery if detected early, however, treatment options for patients with metastatic melanoma are limited and the five-year survival rate for metastatic melanoma had been 15–20% before the advent of immunotherapy. Treatment with immune checkpoint inhibitors has increased long-term survival outcomes in patients with advanced melanoma to as high as 50% although individual response can vary greatly. A mutation within the MAPK pathway leads to uncontrollable growth and ultimately develops into cancer. The most common driver mutation that leads to this characteristic overactivation in the MAPK pathway is the B-RAF mutation. Current combinations of BRAF and MEK inhibitors that have demonstrated improved patient outcomes include dabrafenib with trametinib, vemurafenib with cobimetinib or encorafenib with binimetinib. Treatment with BRAF and MEK inhibitors has met challenges as patient responses began to drop due to the development of resistance to these inhibitors which paved the way for development of immunotherapies and other small molecule inhibitor approaches to address this. Resistance to these inhibitors continues to push the need to expand our understanding of novel mechanisms of resistance associated with treatment therapies. This review focuses on the current landscape of how resistance occurs with the chronic use of BRAF and MEK inhibitors in BRAF-mutant melanoma and progress made in the fields of immunotherapies and other small molecules when used alone or in combination with BRAF and MEK inhibitors to delay or circumvent the onset of resistance for patients with stage III/IV BRAF mutant melanoma.

https://www.mdpi.com/2072-6694/12/2/482/pdf

Current Advances in the Treatment of BRAF-Mutant Melanoma

Recent studies have highlighted a role of HER3 in ER and HER2-driven breast cancers. We sought to investigate the role of patient-derived HER3 mutations in ER+ and HER2+ breast cancer cells using ectopic expression of HER3 mutants. We found that HER3T355I mutant is activating with increased cell proliferation in ER+ T47D and MCF-7 breast cancer cells lacking HER2 over-expression. Immunoblotting and receptor tyrosine kinase array results indicated that T47D and MCF-7 cells expressing HER3T355I had increased p-HER4 and p-HER1 expression. Our data showed that HER3T355I induced cell proliferation is via HER4/HER1-dependent ERK1/2 and cyclinD1 mediated pathways in ER+ cells. ERα expression is upregulated in ER+ cells expressing HER3T355I mutant. We noted crosstalk between ERα and HER3 in T47D cells. Several HER3 mutants (F94L, G284R, D297Y, T355I, and E1261A) acquired a gain-of-function phenotype in MCF10AHER2 cells and were resistant to lapatinib. These mutants increased HER2-HER3 heterodimerization. Knocking down HER3 from ovarian and colorectal cancers with endogenous HER3 mutations abrogated cancer cell proliferation. Overall, this study provides the first systematic assessment of how mutations in HER3 affect response of ER+ and HER2+ breast cancers to clinically relevant inhibitors and finds that HER3 mutations can be activating independent of HER2 over-expression.

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Activating HER3 mutations in breast cancer.

B-Rapidly Accelerated Fibrosarcoma (BRAF) mutations are found in about 50% of melanoma patients. Treatment with Food and Drug Administration (FDA)-approved BRAF and MAP/ERK kinase (MEK) inhibitors has improved progression free and overall survival of patients with BRAF mutant melanoma. However, all responders develop resistance typically within 1 year of treatment with these inhibitors. Evidence indicates that reactive oxygen species (ROS) levels are elevated after BRAF pathway inhibition treatment. We aim to decipher the role of mitochondrial antioxidant proteins relative to ROS levels and BRAF pathway inhibitor resistance. We observed BRAF mutant melanoma cells treated with the combination of a MEK inhibitor (trametinib) and a BRAF inhibitor (dabrafenib), exhibited elevated ROS levels, both in in vitro and in vivo melanoma models. We next generated trametinib- and dabrafenib-resistant (TDR) cells and found increased ROS levels after acquisition of resistance. An immunofluorescence experiment showed an increase of DNA damage in TDR cell lines. Furthermore, we observed that TDR cells increased superoxide dismutase 2 (SOD2), an antioxidant, at both mRNA and protein levels, with the upregulation of the transcription factor Nuclear Factor (NF)-κB. Knockdown of SOD2 significantly reduced the growth of BRAF pathway inhibitor-resistant cells. In addition, the results indicate that TDR cells can be re-sensitized to BRAF pathway inhibitors by the ROS scavenger, N-Acetyl Cysteine (NAC). Overall, these data indicate that BRAF pathway inhibitor-resistant cells can compensate for elevated ROS via increased expression of the antioxidant SOD2.

https://www.mdpi.com/2072-6694/12/6/1661/pdf

BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels.

BRAF mutations occur in about 50% of melanoma patients. FDA approved BRAF and MEK inhibitors have improved the prognosis of patients with BRAF mutations. However, all responders develop resistance typically within one year of treatment. Recent observations demonstrate that BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells. A100, identified from a library screen, is a ROS-activated prodrug that self-cyclizes into a stable bicyclic ring and causes DNA double strand breaks. We proposed to examine if ROS activated therapy will inhibit tumor growth and evade resistance to BRAF inhibitors. In this study, the BRAF inhibitor dabrafenib was used to generate resistant cell lines (A375DR, SK-MEL-24DR and WM-115DR). Flow cytometry experiments showed that ROS levels are increased in these dabrafenib-resistant cells as compared to parental cells, assessed by both the H2DCFDA and MitoSOX assays. Furthermore, we observed that resistant cells had increased levels of the mitochondrial enzymes SOD2 and PRDX1, which function to reduce ROS levels in the mitochondria. We found that A100 sensitized the resistant melanoma cells to dabrafenib and induced DNA damage. Co-treatment of both A100 and dabrafenib significantly suppressed in vitro cell proliferation and three- dimensional (3D) matrigel growth. This study suggests that the combination of A100 with a BRAF inhibitor could be a potential strategy to treat melanoma patients with BRAF mutations.

Long Yuan

Papers

HER3 signaling and targeted therapy in cancer.

Current Advances in the Treatment of BRAF-Mutant Melanoma

Activating HER3 mutations in breast cancer.

BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels.

Utilization of Reactive Oxygen Species Targeted Therapy to Prolong the Efficacy of BRAF Inhibitors in Melanoma.