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Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap—a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed ‘kinetic gene clusters’ whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell–specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune–metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti–PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms.Significance:BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms.This article is highlighted in the In This Issue feature, p. 517

Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.

Machine learning evaluation of immune-metabolic targets in predicting patient response to anti-PD1 and anti-CTLA4 combination therapy.

Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell–specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune–metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti–PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms.Significance:BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms.This article is highlighted in the In This Issue feature, p. 517

We detailed a comparison of BipotentR with competing and alternative approaches in Supplementary notes 1, 2, 6, and 7. Supplementary Note 3-5 describes the in vitro findings of ESRRA, the potential clinical relevance of ESRRA across cancer-types, and the association of tumor activity of ESRRA with immune infiltration.

Primer sequences used in this study

Mutation of the oncogene BRAF is among the most common genetic alterations in melanoma. BRAF inhibitors alone or in combination with MEK inhibitors fail to eradicate the tumor in most patients due to combinations of intrinsic or acquired resistance. Therefore, novel strategies are needed to improve the therapeutic efficacy of BRAF inhibition. We demonstrated that dinaciclib has potent antimelanoma effects by inducing BAK-dependent apoptosis through MCL1 reduction. Contrary to dinaciclib, the inhibitors of BRAF/MEK/CDK4/6 induced apoptosis dominantly through a BAX-dependent mechanism. Although the combination of BRAF and MEK inhibitors did not exhibit additive antimelanoma effects, their combination with dinaciclib synergistically inhibited melanoma growth both in vitro and in vivo. Collectively, our present findings suggest dinaciclib to be an effective complementary drug of BAX-dependent antimelanoma drugs by targeting BAK-mediated apoptosis, and other such rational drug combinations can be determined by identifying complementary drugs activating either BAK or BAX.

Supplementary Figure Legend

Supplemental figures S1-6. Supplementary Figure S1. List of genes in human melanoma 19p13.3 focal deletion region. Supplementary Figure S2. The expression of 19p13.3 genes during the progression of human melanoma from nevus to melanoma based on two independent datasets. Supplementary Figure S3. Identify MCAM by microarray gene set enrichment analysis. Supplementary Figure S4. Expression of Mcam in control and Zbtb7a deleted cells by microarray and real time PCR analysis. Supplementary Figure S5. MCAM copy number variation and prognosis in human melanoma. Supplementary Figure S6. A, luciferase assay was performed to assess the effects of increasing amount of ZBTB7A on the luciferase activity of pGL3-Basic and pGL3-MCAM vector in SK-MEL-28 cells. Data shown are mean {plus minus} s.d. of three experiments. B, expression of FLAG-ZBTB7A protein relative to endogenous ZBTB7A in HeLa cells. C, ChIP and real time PCR to quantify MCAM promoter DNA amount with two additional pairs of primers. Data shown are mean {plus minus} s.d of four experiments. D, sequence alignment between human and mouse MCAM promoter region (-300-+1). Conserved ZBTB7A binding sites in human and mouse MCAM promoter are marked. E, sequence of human MCAM promoter used in PGL3 promoter cloning, and the ZBTB7A binding sites, transcription start site are labeled.

BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8+ T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten−/− syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8+ T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8+ T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy. Cancer Immunol Res; 2(7); 643–54. ©2014 AACR.

PDF file 272K, This file contains an extended description of the materials and methods used to generate steady-state protein expression, drug sensitivity, lysate generation and blotting techniques along with all DNA sequences for loss of function reagents

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations.Significance:Oncogenic G9a abnormalities drive tumorigenesis and the “cold” immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore “hot” tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890

Supplementary Figure 1. BP cells behave like classic BRAFV600E melanomas after BRAF inhibition. Supplementary Figure 2. Subcutaneously implanted BP cells develop into tumors. Supplementary Figure 3. BRAF inhibition is associated with an increase in density of tumor-infiltrating T cells. Supplementary Figure 4. Kinetics of functional CD8 T cell markers. Supplementary Figure 5. BRAF inhibition is associated with an increase in overall PD-L1 and PD-L2 expression in the tumor. Supplementary Figure 6. Cell type specific changes in PD-L1 expression after BRAF inhibitors. Supplementary Figure 7. PD-1 or PD-L1 blockade synergy with BRAF inhibitors is associated with increased density of tumor-infiltrating T cells.

Supplementary Figure 1 shows that CDK2 and CDK9 are involved in BAK-dependent melanoma apoptosis induced by dinaciclib.Supplementary Figure 2 shows that BRAF inhibition suppresses melanoma through BAX. Supplementary Figure 3 shows combinational effects of dinaciclib with BAX-activating drugs.

PDF file 89K, This file contains a table listing the top 75 genes correlated with RAF-inhibitor sensitivity in BRAFV600E-mutant cell lines as well as a table describing the IHC results summarized in Main Figure 2b and 2c

PDF file 615K, This file contains a series of additional experiments and analyses related to Main Figures 1-5, including more extensive transcript/drug sensitivity correlations, mRNA-expression validation experiments,IHC controls, confirmation of drug sensitivity predictions and perturbation experiments

PDF file - 19K, Melanoma antigen expression is decreased at time of progression.

''Temozolomide induced PARP activation deregulates NAD metabolism. Additive cytotoxic effect of TMZ and NAMPT inhibitors in IDH1 mutant cancers. NAD and cell viability effects with TMZ and NAMPT inhibitors in endogenous IDH1 mutant cancers. Combination treatment with TMZ and NAMPT inhibitor in IDH1 mutant and wild-type cells. Cell death mechanisms that underlie combined TMZ and NAMPT inhibitor treatment in IDH1 mutant cells. In vivo effects of combined TMZ and NAMPT inhibitor treatment for endogenous IDH1 mutant cancer.''

PDF file - 349K, Inhibition of BRAFV600E is not associated with a significant increase in HLA expression in patients with metastatic melanoma.

Supplementary Figures and Figure Legends

'Temozolomide induced PARP activation deregulates NAD metabolism. Additive cytotoxic effect of TMZ and NAMPT inhibitors in IDH1 mutant cancers. NAD and cell viability effects with TMZ and NAMPT inhibitors in endogenous IDH1 mutant cancers. Combination treatment with TMZ and NAMPT inhibitor in IDH1 mutant and wild-type cells. Cell death mechanisms that underlie combined TMZ and NAMPT inhibitor treatment in IDH1 mutant cells. In vivo effects of combined TMZ and NAMPT inhibitor treatment for endogenous IDH1 mutant cancer.'

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. Clin Cancer Res; 19(5); 1225–31. ©2013 AACR.

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PDF file - 30K, Inhibition of BRAFV600E increases expression of MDAs in tumors of patients with metastatic melanoma.

PDF file - 112K, Inhibition of BRAFV600E increases expression of MART-1 in tumors of patients with metastatic melanoma.

Purpose: Successful development of targeted therapy combinations for cancer patients depends on first discovering such combinations in predictive preclinical models. Stable cell lines and mouse xenograft models can have genetic and phenotypic drift and may take too long to generate to be useful as a personalized medicine tool.Experimental Design: To overcome these limitations, we have used a platform of ultra-high-throughput functional screening of primary biopsies preserving both cancer and stroma cell populations from melanoma patients to nominate such novel combinations from a library of thousands of drug combinations in a patient-specific manner within days of biopsy. In parallel, patient-derived xenograft (PDX) mouse models were created and novel combinations tested for their ability to shrink matched PDXs.Results: The screening method identifies specific drug combinations in tumor cells with patterns that are distinct from those obtained from stable cell lines. Screening results were highly specific to individual patients. For patients with matched PDX models, we confirmed that individualized novel targeted therapy combinations could inhibit tumor growth. In particular, a combination of multi-kinase and PI3K/Akt inhibitors was effective in some BRAF–wild-type melanomas, and the addition of cediranib to the BRAF inhibitor PLX4720 was effective in a PDX model with BRAF mutation.Conclusions: This proof-of-concept study demonstrates the feasibility of using primary biopsies directly for combinatorial drug discovery, complementing stable cell lines and xenografts, but with much greater speed and efficiency. This process could potentially be used in a clinical setting to rapidly identify therapeutic strategies for individual patients. Clin Cancer Res; 23(16); 4680–92. ©2017 AACR.

PDF file - 50K, Patient Characteristics.

Screening results for drug combinations, expressed as percent of control (DMSO-treated) values.

Supplementary Methods, Figure Legends 1-6 from An Oncogenic Role for ETV1 in Melanoma

Supplementary Table 1 and Figures 1-4 from TFE3 Fusions Activate MET Signaling by Transcriptional Up-regulation, Defining Another Class of Tumors as Candidates for Therapeutic MET Inhibition

Supplementary Figure 5 from Selective BRAFV600E Inhibition Enhances T-Cell Recognition of Melanoma without Affecting Lymphocyte Function

Supplementary Methods and Figure Legends 1-4 from TFE3 Fusions Activate MET Signaling by Transcriptional Up-regulation, Defining Another Class of Tumors as Candidates for Therapeutic MET Inhibition

Clear cell sarcoma (CCS), a childhood tumor of the tendons and aponeuroses, is uniformly fatal once it has metastasized because of its profound therapeutic resistance. CCS is characterized by production of a chimeric transcription factor, EWS-ATF1, which is formed as the result of a disease-specific chromosomal translocation. EWS-ATF1 activates the melanocyte transcription factor MITF, which in turn activates transcription of c-Met, an oncogenic receptor tyrosine kinase recently shown to be activated in CCS. Based on this connection, we hypothesized that c-Met inhibition may offer a strategy to treat CCS, as an indirect tactic to defeat a transforming pathway downstream of EWS-ATF1. Here, we show that primary CCS and CCS-derived cell lines express c-Met, which is activated in an autocrine fashion by its ligand hepatocyte growth factor (HGF)/scatter factor in some CCS cell lines. c-Met expression is critical for CCS invasion, chemotaxis, and survival. Blocking c-Met activity with a small-molecule inhibitor (SU11274) or a neutralizing antibody to its ligand HGF (AMG 102) significantly reduced CCS cell growth in culture. Similarly, AMG 102 significantly suppressed in vivo tumor growth in an autocrine xenograft model of CCS. Collectively, these findings suggest the HGF:c-Met signaling axis as a candidate therapeutic target to improve clinical management of CCS. Cancer Res; 70(2); 639–45

Supplementary Figure 1 from Identification of the Receptor Tyrosine Kinase c-Met and Its Ligand, Hepatocyte Growth Factor, as Therapeutic Targets in Clear Cell Sarcoma