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Blocking cancer genomic instability may prevent tumor diversification and escape from therapies. We show that, after MAPK inhibitor (MAPKi) therapy in patients and mice bearing patient-derived xenografts (PDX), acquired resistant genomes of metastatic cutaneous melanoma specifically amplify resistance-driver, nonhomologous end-joining (NHEJ), and homologous recombination repair (HRR) genes via complex genomic rearrangements (CGR) and extrachromosomal DNAs (ecDNA). Almost all sensitive and acquired-resistant genomes harbor pervasive chromothriptic regions with disproportionately high mutational burdens and significant overlaps with ecDNA and CGR spans. Recurrently, somatic mutations within ecDNA and CGR amplicons enrich for HRR signatures, particularly within acquired resistant tumors. Regardless of sensitivity or resistance, breakpoint–junctional sequence analysis suggests NHEJ as critical to double-stranded DNA break repair underlying CGR and ecDNA formation. In human melanoma cell lines and PDXs, NHEJ targeting by a DNA-PKCS inhibitor prevents/delays acquired MAPKi resistance by reducing the size of ecDNAs and CGRs early on combination treatment. Thus, targeting the causes of genomic instability prevents acquired resistance. Significance: Acquired resistance often results in heterogeneous, redundant survival mechanisms, which challenge strategies aimed at reversing resistance. Acquired-resistant melanomas recurrently evolve resistance-driving and resistance-specific amplicons via ecDNAs and CGRs, thereby nominating chromothripsis–ecDNA–CGR biogenesis as a resistance-preventive target. Specifically, targeting DNA-PKCS/NHEJ prevents resistance by suppressing ecDNA/CGR rearrangements in MAPKi-treated melanomas. This article is highlighted in the In This Issue feature, p. 799

Metastasis and failure of present-day therapies represent the most common causes of mortality in patients with cutaneous melanoma. To identify the underlying genetic and transcriptomic landscapes, in this study we analyzed multi-organ metastases and tumor-adjacent tissues from 11 rapid autopsies after treatment with MAPK inhibitor (MAPKi) and/or immune checkpoint blockade (ICB) and death due to acquired resistance. Either treatment elicits shared genetic alterations that suggest immune-evasive, cross-therapy resistance mechanisms. Large, non-clustered deletions, inversions and inter-chromosomal translocations dominate rearrangements. Analyzing data from separate melanoma cohorts including 345 therapy-naive patients and 35 patients with patient-matched pre-treatment and post-acquired resistance tumor samples, we performed cross-cohort analyses to identify MAPKi and ICB as respective contributors to gene amplifications and deletions enriched in autopsy versus therapy-naive tumors. In the autopsy cohort, private/late mutations and structural variants display shifted mutational and rearrangement signatures, with MAPKi specifically selecting for signatures of defective homologous-recombination, mismatch and base-excision repair. Transcriptomic signatures and crosstalks with tumor-adjacent macroenvironments nominated organ-specific adaptive pathways. An immune-desert, CD8+-macrophage-biased archetype, T-cell exhaustion and type-2 immunity characterized the immune contexture. This multi-organ analysis of therapy-resistant melanoma presents preliminary insights with potential to improve therapeutic strategies.

MAPK inhibitor (MAPKi) therapy in melanoma leads to the accumulation of tumor-surface PD-L1/L2, which may evade antitumor immunity and accelerate acquired resistance. Here, we discover that the E3 ligase ITCH binds, ubiquitinates, and downregulates tumor-surface PD-L1/L2 in MAPKi-treated human melanoma cells, thereby promoting T-cell activation. During MAPKi therapy in vivo, melanoma cell–intrinsic ITCH knockdown induced tumor-surface PD-L1, reduced intratumoral cytolytic CD8+ T cells, and accelerated acquired resistance only in immune-competent mice. Conversely, tumor cell–intrinsic ITCH overexpression reduced MAPKi-elicited PD-L1 accumulation, augmented intratumoral cytolytic CD8+ T cells, and suppressed acquired resistance in BrafV600MUT, NrasMUT, or Nf1MUT melanoma and KrasMUT-driven cancers. CD8+ T-cell depletion and tumor cell–intrinsic PD-L1 overexpression nullified the phenotype of ITCH overexpression, thereby supporting an in vivo ITCH–PD-L1–T-cell regulatory axis. Moreover, we identify a small-molecular ITCH activator that suppresses acquired MAPKi resistance in vivo. Thus, MAPKi-induced PD-L1 accelerates resistance, and a PD-L1–degrading ITCH activator prolongs antitumor response.Significance:MAPKi induces tumor cell–surface PD-L1 accumulation, which promotes immune evasion and therapy resistance. ITCH degrades PD-L1, optimizing antitumor T-cell immunity. We propose degrading tumor cell–surface PD-L1 and/or activating tumor-intrinsic ITCH as strategies to overcome MAPKi resistance.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Figure S1. Associations of CGRs and ecDNAs with ploidy, expression, and enhancers. Supplementary Figure S2. Analysis of chromothripsis, ecDNAs, and CGRs in MAPKi-sensitive/-naïve versus -resistant melanoma. Supplementary Figure S3. Inferring double-stranded DNA break repair pathways underlying ecDNA and CGR breakpoint junctions. Supplementary Figure S4. Single-agent inhibitory potencies in human melanoma, PDAC, and NSCLC clonogenic growth assays. Supplementary Figure S5. DNA-PKi and/or PARPi co-treatment prevents acquired MAPKi-resistance in human melanoma cell lines. Supplementary Figure S6. In vivo impacts of DNA-PKi when combined with MAPKi.

Supplementary Figure from Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired Resistance

Blocking cancer genomic instability may prevent tumor diversification and escape from therapies. We show that, after MAPK inhibitor (MAPKi) therapy in patients and mice bearing patient-derived xenografts (PDX), acquired resistant genomes of metastatic cutaneous melanoma specifically amplify resistance-driver, nonhomologous end-joining (NHEJ), and homologous recombination repair (HRR) genes via complex genomic rearrangements (CGR) and extrachromosomal DNAs (ecDNA). Almost all sensitive and acquired-resistant genomes harbor pervasive chromothriptic regions with disproportionately high mutational burdens and significant overlaps with ecDNA and CGR spans. Recurrently, somatic mutations within ecDNA and CGR amplicons enrich for HRR signatures, particularly within acquired resistant tumors. Regardless of sensitivity or resistance, breakpoint–junctional sequence analysis suggests NHEJ as critical to double-stranded DNA break repair underlying CGR and ecDNA formation. In human melanoma cell lines and PDXs, NHEJ targeting by a DNA-PKCS inhibitor prevents/delays acquired MAPKi resistance by reducing the size of ecDNAs and CGRs early on combination treatment. Thus, targeting the causes of genomic instability prevents acquired resistance.Significance:Acquired resistance often results in heterogeneous, redundant survival mechanisms, which challenge strategies aimed at reversing resistance. Acquired-resistant melanomas recurrently evolve resistance-driving and resistance-specific amplicons via ecDNAs and CGRs, thereby nominating chromothripsis–ecDNA–CGR biogenesis as a resistance-preventive target. Specifically, targeting DNA-PKCS/NHEJ prevents resistance by suppressing ecDNA/CGR rearrangements in MAPKi-treated melanomas.This article is highlighted in the In This Issue feature, p. 799

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Supplementary Figures, Figure Legends, and Table Legends

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAFnon-V600, and BRAFV600 mutations. Tumor cell–intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti–PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy.Significance:Type I RAFi + MEKi are indicated only in certain BRAFV600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521

Table S1: Clinical characteristics of patients who donated MAPKi-sensitive/-naive and acquired MAPKi-resistant melanoma. Table S2: Genomic alterations, chromothripsis, and ecDNA-/CGR-amplicons detected in MAPKi-sensitive/-naive and acquired MAPKi-resistant melanoma genomes. Table S3: Genes amplified by ecDNAs, CGRs, and linear amplicons along with copy numbers in MAPKi-sensitive/-naïve and acquired MAPKi-resistant melanoma genomes. Table S4: Co-occurrence of resistance-associated genetic alterations (identified in prior studies) with resistance-driver ecDNA and CGR amplicons (identified in the current study). Table S5: Normalized RNA-seq-based transcript expression in MAPKi-sensitive/-naïve and acquired-resistant tumors. Table S6: MAPK-reactivation amplicons in acquired resistance, copy numbers, and amplicon sub-types. Table S7: MAPKi-resistant genes amplified by ecDNAs and CGRs utilized for pathway enrichment analysis. Table S8: Enhancers associated with ecDNAs and CGRs in MAPKi-sensitive/-naive and acquired MAPKi-resistant melanoma genomes. Table S9: Frequencies of enriched SBS signatures reflecting defective DNA repair mechanisms within chromothripsis and non-chromothriptic regions in MAPKi-sensitive (n = 16) and acquired-resistant (n = 31) tumors. Table S10: ecDNAs, CGRs, copy numbers, and associated genes in human melanoma cell lines (M229, M249) on vehicle treatment and early on treatment. Table S11: ecDNAs, CGRs, copy numbers, associated genes in vehicle-treated and early on-treatment PDX tumors.

MAPK inhibitor (MAPKi) therapy in melanoma leads to the accumulation of tumor-surface PD-L1/L2, which may evade antitumor immunity and accelerate acquired resistance. Here, we discover that the E3 ligase ITCH binds, ubiquitinates, and downregulates tumor-surface PD-L1/L2 in MAPKi-treated human melanoma cells, thereby promoting T-cell activation. During MAPKi therapy in vivo, melanoma cell–intrinsic ITCH knockdown induced tumor-surface PD-L1, reduced intratumoral cytolytic CD8+ T cells, and accelerated acquired resistance only in immune-competent mice. Conversely, tumor cell–intrinsic ITCH overexpression reduced MAPKi-elicited PD-L1 accumulation, augmented intratumoral cytolytic CD8+ T cells, and suppressed acquired resistance in BrafV600MUT, NrasMUT, or Nf1MUT melanoma and KrasMUT-driven cancers. CD8+ T-cell depletion and tumor cell–intrinsic PD-L1 overexpression nullified the phenotype of ITCH overexpression, thereby supporting an in vivo ITCH–PD-L1–T-cell regulatory axis. Moreover, we identify a small-molecular ITCH activator that suppresses acquired MAPKi resistance in vivo. Thus, MAPKi-induced PD-L1 accelerates resistance, and a PD-L1–degrading ITCH activator prolongs antitumor response. Significance: MAPKi induces tumor cell–surface PD-L1 accumulation, which promotes immune evasion and therapy resistance. ITCH degrades PD-L1, optimizing antitumor T-cell immunity. We propose degrading tumor cell–surface PD-L1 and/or activating tumor-intrinsic ITCH as strategies to overcome MAPKi resistance. This article is highlighted in the In This Issue feature, p. 1825

Background The concept of multi-step progression from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma (ADC) has been proposed, and ground-glass nodules (GGNs) may play a critical role during the early lung tumorigenesis. We present the first comprehensive description of the genomic architecture of GGNs to unravel the genetic basis of GGN. Methods We investigated 30 GGN-like lungs ADC by performing >1,000× whole-exome sequencing (WES) and characterized the genomic variations and evaluate the relationship between the clinicopathologic and molecular characteristics in this disease. Results Despite the low somatic mutation burden, GGNs exhibited high intratumor heterogeneity (ITH) characterized by the proportion of subclonal mutations. Different mutagenesis shaped the genomes of GGN during cancer evolution and were mostly featured by molecular clock-like signatures that occur in clonal mutations and defective DNA mismatch signatures that occur in subclonal mutations. Moreover, 10.7-67.1% clonal mutations occurred after whole-genome doubling (WGD), indicating that WGD could be a frequent truncal event in GGNs. Samples with WGD showed higher genomic instability but lower ITH. These GGNs were characterized by recurrent focal copy-number changes that are highly associated with tumorigenesis, with only two genes (EGFR and RBM10) that were recurrently mutated. Additionally, GGNs with different pathological subtypes or computed tomography (CT) features exhibited distinct genetic characteristics. Lepidic predominant or pure GGNs in CT images carried a lower mutation burden and had a relatively stable genome than nonlepidic or mixed GGNs. GGNs with RBM10 mutations tended to accompany a pathologically lepidic pattern, indicating RBM10 may drive the distinct subtype of lung cancer with better prognosis. Conclusions These findings facilitated interpreting the genomic characteristics of GGNs, provided insight into the early stages of lung cancer evolution, and possessed potential clinical significance.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAFnon-V600, and BRAFV600 mutations. Tumor cell–intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti–PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. Significance: Type I RAFi + MEKi are indicated only in certain BRAFV600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited. This article is highlighted in the In This Issue feature, p. 521

Background: In cutaneous melanoma, the burden of chromothripsis is high prior to targeted therapy, and additional chromothripsis appears to be a key evolutionary mechanism by which cancer rapidly generates and accumulates highly dynamic structural variants (SVs). Blocking cancer genomic instability may prevent tumor escape from targeted therapies. Methods: We assembled three cohorts of tissues for WGS-based analysis of SVs. The first cohort consisted of patient-matched normal tissues, BRAF V600MUT melanoma tumors before MAPKi therapy and at disease progression (n=10 normal tissues; n=10 pretreatment tumors; n=17 acquired-resistant tumors; n=10 patients). The second cohort consisted of rapid autopsy melanoma tissues (n=3 normal tissues; n=12 acquired-resistant tumors; n=6 metastatic organ sites). The third cohort consisted of cutaneous PDX tumors. To study acquired MAPKi-resistance at the whole-genome level, we subjected PDXs (n=6 models; 1 BRAF MUT and 5 NRAS MUT models) to MAPKi therapy in NSG mice at doses sufficient to elicit tumor regression, and then generated acquired MAPKi-resistant tumors. In total, we used vehicle-treated tumors (n=6), acquired-resistant tumors (n=12), and patient-matched normal tissues (n=6) to generate WGS data. Results: Analysis of genomic amplicons due to intrachromosomal complex genomic rearrangements (CGRs) and extrachromosomal circular DNAs (ecDNAs) uncovered a significant (unpaired Student’s t-test, p=0.0002) association between acquired-resistant tumors and CGRs and/or ecDNAs harboring bona fide MAPKi-resistance genes and revealed copy number amplification of BRAF (range 4.5-27), NRAS (range 5-13), HRAS (range 13-16), MYC (range 12-15) and EGFR (CN 4.6-5), known to drive acquired MAPKi-resistance. Moreover, we validated a recurrent ecDNA by direct isolation and high-depth sequencing using a new approach referred to as CRISPR-CATCH. This alternative technique confirmed the circularized junctions of a 890 kb, driver ecDNA within this acquired-resistant clinical tumor sample. Additionally, resistance-specific (versus sensitivity-specific) chromothriptic single-base substitutions (SBSs) enriched for signatures of defects in base excision repair (BER) and in DNA mismatch repair (MMR) (Wilcoxon rank sum test, p=0.04 and p=0.005 respectively) in 14 of 31 resistant tumors (10 of 16 patients). Moreover, breakpoint-sequence analysis inferred non-homologous end-joining (NHEJ) as critical, and homologous recombination repair (HRR) as adjunctive, to DNA double-stranded break repair underlying CGR and ecDNA formation harboring MAPK-reactivation or MAPKi resistance-driver genes. Inhibition of DNA-PKc or PARP1/2, even only initially during MAPKi treatment, suppressed acquired MAPKi-resistance in melanoma cell lines (BRAF V600MUT n=3 and NRAS Q61MUT n=3) and blunted the expansion of ecDNA + CGR genomic spans. In vivo, DNA-PKi in combination with MAPKi forestalled resistance, reduced ecDNA and CGRs size, and suppressed the contribution of NHEJ in 5 out of 5 cutaneous melanoma PDXs analyzed. Conclusions: Our findings advance the concepts that preventing—instead of reversing—acquired resistant phenotypes may be more clinically impactful and that targeting DNA-PKCS and NHEJ lies at the center of this approach in stabilizing cancer genomes during oncogene-targeted therapies. Citation Format: Prashanthi Dharanipragada, Xiao Zhang, Sixue Liu, Shirley H. Lomeli, Aayoung Hong, Yan Wang, Zhentao Yang, Agustin Vega-Crespo, Antoni Ribas, Stergios J. Moschos, Gatien Moriceau, Roger S. Lo. Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB251.

V2O5 is a promising candidate for cathode active material for Li-ion batteries due to its high theoretical specific capacity but suffers from poor rate capability and cycling stability. To cover these disadvantages, in this work, a low-cost and facile sol-gel method to prepare TiO2-coated V2O5 microspheres is developed for the first time. The prepared V2O5@TiO2 composite could deliver an initial capacity of 297.7 mAh g−1 at a current density of 100 mA g−1 in the potential range of 2.0–4.0 V (vs. Li+/Li). Moreover, the capacity of 247.0 mA h g−1 could be delivered at 1000 mA g−1, and 86% of capacity could be retained after 100 cycles. Even at a large current density of 5000 mA g−1, it could still deliver a high capacity of 197.3 mA h g−1 with a capacity retention of 93.5% after 200 cycles. The outstanding rate and cycling stability of V2O5@TiO2 composite indicate that it holds bright prospect for using as an excellent cathode material for rechargeable lithium batteries.

Uncovering the functionally essential variations related to tumorigenesis and tumor progression from cancer genomics data is still challenging due to the genetic diversity among patients, and extensive inter- and intra-tumoral heterogeneity at different levels of gene expression regulation, including but not limited to the genomic, epigenomic, and transcriptional levels. To minimize the impact of germline genetic heterogeneities, in this study, we establish multiple primary cultures from the primary and recurrent tumors of a single patient with hepatocellular carcinoma (HCC). Multi-omics sequencing was performed for these cultures that encompass the diversity of tumor cells from the same patient. Variations in the genome sequence, epigenetic modification, and gene expression are used to infer the phylogenetic relationships of these cell cultures. We find the discrepancy among the relationships revealed by single nucleotide variations (SNVs) and transcriptional/epigenomic profiles from the cell cultures. We fail to find overlap between sample-specific mutated genes and differentially expressed genes (DEGs), suggesting that most of the heterogeneous SNVs among tumor stages or lineages of the patient are functionally insignificant. Moreover, copy number alterations (CNAs) and DNA methylation variation within gene bodies, rather than promoters, are significantly correlated with gene expression variability among these cell cultures. Pathway analysis of CNA/DNA methylation-related genes indicates that a single cell clone from the recurrent tumor exhibits distinct cellular characteristics and tumorigenicity, and such an observation is further confirmed by cellular experiments both in vitro and in vivo. Our systematic analysis reveals that CNAs and epigenomic changes, rather than SNVs, are more likely to contribute to the phenotypic diversity among subpopulations in the tumor. These findings suggest that new therapeutic strategies targeting gene dosage and epigenetic modification should be considered in personalized cancer medicine. This culture model may be applied to the further identification of plausible determinants of cancer metastasis and relapse.

Spatial genetic and phenotypic diversity within solid tumors has been well documented. Nevertheless, how this heterogeneity affects temporal dynamics of tumorigenesis has not been rigorously examined because solid tumors do not evolve as the standard population genetic model due to the spatial constraint. We therefore, propose a neutral spatial (NS) model whereby the mutation accumulation increases toward the periphery; the genealogical relationship is spatially determined and the selection efficacy is blunted (due to kin competition). In this model, neutral mutations are accrued and spatially distributed in manners different from those of advantageous mutations. Importantly, the distinctions could be blurred in the conventional model. To test the NS model, we performed a three-dimensional multiple microsampling of two hepatocellular carcinomas. Whole-genome sequencing (WGS) revealed a 2-fold increase in mutations going from the center to the periphery. The operation of natural selection can then be tested by examining the spatially determined clonal relationships and the clonal sizes. Due to limited migration, only the expansion of highly advantageous clones can sweep through a large part of the tumor to reveal the selective advantages. Hence, even multiregional sampling can only reveal a fraction of fitness differences in solid tumors. Our results suggest that the NS patterns are crucial for testing the influence of natural selection during tumorigenesis, especially for small solid tumors.

Summary Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer., Graphical abstract, Highlights • FLT4 nsSNVs favor response; CDKN2A nsSNVs favor non-response; high TMB improves RFS • Recurrences select for CN loss in PTEN, JAK2 and/or gain in YAP1, MDM2, PPARG • T cell clones (blood) diversify and preexisting clones (tumors) expand with response • High ratios of TREG/Th17 in pretreatment blood predict innate resistance, Liu et al. nominate multi-omic correlates of response, recurrence, and survival in individuals treated with neoadjuvant anti-PD-1 therapy for resectable, locally advanced, oral cavity SCC. Future validation of blood- and tumor-based biomarkers and mechanistic insights have implications for neoadjuvant and adjuvant management of individuals with systemic treatment-naive resectable disease.

β-adrenoreceptors do not inhibit sympathetic vasoconstriction in resting or contracting muscle of male or female rats, regardless of training status. Sympatholysis was enhanced in female, compared to male rats; however, β-adrenoreceptors were not responsible for the enhanced sympatholysis. These findings indicate that β-adrenoreceptors do not contribute to the regulation of sympathetic vasoconstriction in resting and contracting skeletal muscle and suggest that β-adrenoreceptors do not underlie sex differences in the neural control of the circulation.

Classical r- vs. K-selection theory describes the trade-offs between high reproductive output and competitiveness and guides research in evolutionary ecology. While its impact has waned in the recent past, cancer evolution may rekindle it. Herein, we impose r- or K-selection on cancer cell lines to obtain strongly proliferative r cells and highly competitive K cells to test ideas on life-history strategy evolution. RNA-seq indicates that the trade-offs are associated with distinct expression of genes involved in the cell cycle, adhesion, apoptosis, and contact inhibition. Both empirical observations and simulations based on an ecological competition model show that the trade-off between cell proliferation and competitiveness can evolve adaptively. When the r and K cells are mixed, they exhibit strikingly different spatial and temporal distributions. Due to this niche separation, the fitness of the entire tumor increases. The contrasting selective pressure may operate in a realistic ecological setting of actual tumors.

SPRED1 is a negative regulator of the MAPK pathway frequently deleted in human melanoma. Through in vivo modeling in zebrafish and mechanistic analyses in human cell lines, Ablain et al. demonstrate that SPRED1 inactivation confers resistance to targeted inhibition of V600 mutant BRAF, the most common driver of melanoma., Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma.

Xanthoceras sorbifolium Bunge is a potential biofuel plant, owing to its high seed oil content. But planted forest experiments and practice indicate that field management before flowering played a vital role in increasing the female to male ration and further raise seed yield. Thus, knowing the onset of flowering in advance is crucial for taking management measures to regulate floral development. To predict the first flowering date, we developed a predictive model with linear regression, support vector machine, and random forest models. Mean monthly temperatures with chilling and forcing accumulation (November–March) and preseason temperatures one month prior to the onset of flowering were taken as independent variables. Chilling accumulation was calculated using chilling portion (CP) with the Dynamic Model and chilling hours (CH) for the Utah Model, while forcing accumulation was derived from the Growing Degree Hours (GDH) Model. Internal leave-one-out cross validation and external validation were used to examine model performance. Generally, multivariate models had similar errors and performed better than the simple linear regression models. The lowest external and internal error values (3.8 and 2.59 days, respectively) were obtained using GDH with the multivariate linear model for March and CP for November. Regarding practicality, we recommend the use of the linear regression model for CP data from January (external error = 5.7 days, internal error = 5.64 days). The non-significant correlation between one month pre-season temperatures and first flowering date indicates that flowering time is primarily determined by earlier temperature accumulation. The significant influence of chilling accumulation in January on the onset of flowering are in coincidence with that X. sorbifolium is an early flowering species. The stable external errors related to the use of GDH for March data and CP for November data implies that the combination of forcing and chilling accumulation plays a critical role in initiating flowering onset. Our study provides a basis for forecasting flowering onset and regulating the floral development of X. sorbifolium.

The aim of this study is to investigate the role of Hepatitis B virus x (HBx) in the growth and secretion of human placental trophoblasts. Firstly, placenta tissues were collected from pregnant HBV carriers with various viral loads. The results of immunohistochemical technique showed that the HBx protein and pEGFR protein levels were both markedly increased with the viral load elevation. Then, a placental trophoblast cell strain (JEG-3-HBx), which stably expressed HBx mRNA and protein, was established with the pcDNA-HBx transfection followed by the G418 selection. The JEG-3-HBx strain displayed distinct activation of the EGFR/AKT pathway, a lower level of cell apoptosis, and higher secretion levels of placental hormones, including human chorionic gonadotropin (hCG), progesterone, estrogen and β-endorphin. Subsequently, HBx siRNA was used to silence the HBx gene in the JEG-3-HBx strain. Our data showed that the HBx siRNA transfection markedly suppressed the activation of the EGFR/AKT pathway, promoted cell apoptosis, and reduced the secretion of the placental hormones. Finally, EGF was applied to simulate the JEG-3-HBx strain with or without the HBx siRNA transfection. EGF treatment counteracted the reduction of cell apoptosis and the suppression of hormone secretion caused by HBx siRNA in the cell strain. In conclusion, the pEGFR protein was robustly upregulated in HBx-infected human placenta tissues and trophoblast cells. HBx reduces cell apoptosis and promotes the secretion of placental hormones in human placental trophoblast cells via activation of the EGFR/Akt pathway.

Rationally sequencing and combining PD-1/L1-and MAPK-targeted therapies may overcome innate and acquired resistance. Since increased clinical benefit of MAPK inhibitors (MAPKi) is associated with previous immune checkpoint therapy, we compare the efficacies of sequential and/or combinatorial regimens in subcutaneous murine models of melanoma driven by BrafV600, Nras, or Nf1 mutations as well as colorectal and pancreatic carcinoma driven by KrasG12C. Anti-PD-1/L1 lead-in preceding MAPKi combination optimizes response durability by promoting pro-inflammatory polarization of macrophages and clonal expansion of interferon-γhi, and CD8+ cytotoxic and proliferative (versus CD4+ regulatory) Tcells that highly express activation genes. Since therapeutic resistance of melanoma brain metastasis (MBM) limits patient survival, we demonstrate that sequencing anti-PD-1/L1 therapy before MAPKi combination suppresses MBM and improves mouse survival with robust Tcell clonal expansion in both intracranial and extracranial metastatic sites. We propose clinically testing brief anti-PD-1/L1 (± anti-CTLA-4) dosing before MAPKi co-treatment to suppress therapeutic resistance.

Summary Oral cavity squamous cell carcinoma (OCSCC) is a prevalent surgically treated subset of head and neck cancer with frequent recurrence and poor survival. Immunotherapy has demonstrated efficacy in recurrent/metastatic head and neck cancer. However, whether antitumor responses could be fostered by neoadjuvant presurgical immunotherapy remains unclear. Using a Simon’s two-stage design, we present results of a single-arm phase-II trial where 12 patients with stage II-IVA OCSCC received 3 to 4 biweekly doses of 3 mg/kg nivolumab followed by definitive surgical resection with curative intent. Presurgical nivolumab therapy in this cohort shows an overall response rate of 33% (n = 4 patients; 95% CI: 12%–53%). With a median follow up of 2.23 years, 10 out of 12 treated patients remain alive. Neoadjuvant nivolumab is safe, well-tolerated, and is not associated with delays in definitive surgical treatment in this study. This work demonstrates feasibility and safety for incorporation of nivolumab in the neoadjuvant setting for OCSCC (ClinicalTrials.gov: NCT03021993)., Graphical abstract, Highlights • Presurgical nivolumab in patients with resectable oral cancer has an ORR of 33% • 10 out of 12 treated patients remain alive with a median follow up of 2.23 years • Neoadjuvant nivolumab is safe, well-tolerated, and not associated with treatment delays • This shows feasibility of nivolumab in the neoadjuvant setting for OCSCC (NCT03021993), Oral cavity squamous cell carcinoma is a prevalent subset of head and neck cancer with high recurrence rates and poor survival outcomes despite complex multimodality therapy. The current study by Knochelmann et al. demonstrates that presurgical nivolumab therapy has an overall response rate of 33% in this patient population.

Although intratumor diversity driven by selection has been the prevailing view in cancer biology, recent population genetic analyses have been unable to reject the neutral interpretation. As the power to reject neutrality in tumors is often low, it will be desirable to have an alternative means to test selection directly. Here, we utilize gene expression data as a surrogate for functional significance in intra- and intertumor comparisons. The expression divergence between samples known to be driven by selection (e.g., between tumor and normal tissues) is always higher than the divergence between normal samples, which should be close to the neutral level of divergence. In contrast, the expression differentiation between regions of the same tumor, being lower than the neutral divergence, is incompatible with the hypothesis of selectively driven divergence. To further test the hypothesis of neutral evolution, we select a hepatocellular carcinoma tumor that has large intratumor SNV and CNV (single nucleotide variation and copy number variation, respectively) diversity. This tumor enables us to calibrate the level of expression divergence against that of genetic divergence. We observe that intratumor divergence in gene expression profile lags far behind genetic divergence, indicating insufficient phenotypic differences for selection to operate. All these expression analyses corroborate that natural selection does not operate effectively within tumors, supporting recent interpretations of within-tumor diversity. As the expected level of genetic diversity, hence the potential for drug resistance, would be much higher under neutrality than under selection, the issue is of both theoretical and clinical significance.

A high frequency pulse tube cryocooler with precooling (HPTCP) has been developed and tested to meet the requirement of weak magnetic signals measurement, and the performance characteristics are presented in this article. The HPTCP is a two-stage pulse tube cryocooler with the precooling-stage replaced by liquid nitrogen. Two regenerators completely filled with stainless steel (SS) meshes are used in the cooler. Together with cold inertance tubes and cold gas reservoir, a cold double-inlet configuration is used to control the phase relationship of the HPTCP. The experimental result shows that the cold double-inlet configuration has improved the performance of the cooler obviously. The effects of operation parameters on the performance of the cooler are also studied. With a precooling temperature of 78.5 K, the maximum refrigeration capacity is 0.26 W at 15 K and 0.92 W at 20 K when the input electric power are 174 W and 248 W respectively, and the minimum no-load temperature obtained is 10.3 K, which is a new record on refrigeration temperature for high frequency pulse tube cryocooler reported with SS completely used as regenerative matrix.

With the development of high-throughput genomic analysis, sequencing a mouse primary cancer model provides a new opportunity to understand fundamental mechanisms of tumorigenesis and progression. Here, we characterized the genomic variations in a hepatitis-related primary hepatocellular carcinoma (HCC) mouse model. A total of 12 tumor sections and four adjacent non-tumor tissues from four mice were used for whole exome and/or whole genome sequencing and validation of genotyping. The functions of the mutated genes in tumorigenesis were studied by analyzing their mutation frequency and expression in clinical HCC samples. A total of 46 single nucleotide variations (SNVs) were detected within coding regions. All SNVs were only validated in the sequencing samples, except the Hras mutation, which was shared by three tumors in the M1 mouse. However, the mutated allele frequency varied from high (0.4) to low (0.1), and low frequency (0.1-0.2) mutations existed in almost every tumor. Together with a diploid karyotype and an equal distribution pattern of these SNVs within the tumor, these results suggest the existence of subclones within tumors. A total of 26 mutated genes were mapped to 17 terms describing different molecular and cellular functions. All 41 human homologs of the mutated genes were mutated in the clinical samples, and some mutations were associated with clinical outcomes, suggesting a high probability of cancer driver genes in the spontaneous tumors of the mouse model. Genomic sequencing shows that a few mutations can drive the independent origin of primary liver tumors and reveals high heterogeneity among tumors in the early stage of hepatitis-related primary hepatocellular carcinoma.

The aim of this study is to investigate the role of Hepatitis B virus x (HBx) in the growth and secretion of human placental trophoblasts. Firstly, placenta tissues were collected from pregnant HBV carriers with various viral loads. The results of immunohistochemical technique showed that the HBx protein and pEGFR protein levels were both markedly increased with the viral load elevation. Then, a placental trophoblast cell strain (JEG-3-HBx), which stably expressed HBx mRNA and protein, was established with the pcDNA-HBx transfection followed by the G418 selection. The JEG-3-HBx strain displayed distinct activation of the EGFR/AKT pathway, a lower level of cell apoptosis, and higher secretion levels of placental hormones, including human chorionic gonadotropin (hCG), progesterone, estrogen and β-endorphin. Subsequently, HBx siRNA was used to silence the HBx gene in the JEG-3-HBx strain. Our data showed that the HBx siRNA transfection markedly suppressed the activation of the EGFR/AKT pathway, promoted cell apoptosis, and reduced the secretion of the placental hormones. Finally, EGF was applied to simulate the JEG-3-HBx strain with or without the HBx siRNA transfection. EGF treatment counteracted the reduction of cell apoptosis and the suppression of hormone secretion caused by HBx siRNA in the cell strain. In conclusion, the pEGFR protein was robustly upregulated in HBx-infected human placenta tissues and trophoblast cells. HBx reduces cell apoptosis and promotes the secretion of placental hormones in human placental trophoblast cells via activation of the EGFR/Akt pathway.

We analyze to what extent the random SAT and Max-SAT problems differ in their properties. Our findings suggest that for random k-CNF with ratio in a certain range, Max-SAT can be solved by any SAT algorithm with subexponential slowdown, while for formulae with ratios greater than some constant, algorithms under the random walk framework require substantially different heuristics. In light of these results, we propose a novel probabilistic approach for random Max-SAT called ProMS. Experimental results illustrate that ProMS outperforms many state-of-the-art local search solvers on random Max-SAT benchmarks.

To uncover the functionally essential variations related to tumorigenesis and tumor progression from large amount of cancer genomics data is still challenging due to the genetic diversity between patients, and extensive inter- and intra-tumor heterogeneity at the genomic, epigenomic, and transcription levels. Multiple primary cultures derived from phenotypically distinct subpopulations of a single patient may not only represent the diversity and heterogeneity of tumor cell types and states (such as metastasis and drug-resistant), but also narrow the analysis in genomic basis of phenotypic diversity down to the relatively small number of variations between subpopulations with the same genetic background. Performing multi-omics sequencing, we identified variations in single nucleotide, copy number, DNA methylation and gene expression among four primary cell cultures from primary and recurrent tumors in a hepatocellular carcinoma patient. We observed the discrepancy between the phylogenetic relationships revealed by single nucleotide variations (SNVs) and transcriptional profiles in the four cell cultures. The differences in gene expression, which indicated the distinct cellular characteristics and tumorigenic capability in one of the two recurrent cell clones, were correlated to the copy number alterations (CNAs) and DNA methylation variation in gene body and confirmed by the characterization of cellular phenotype. Our multi-omics analysis in heterogeneous clones derived from an individual revealed that CNAs and epigenomic changes, rather than SNVs, dominantly contribute to the phenotypic diversity among subpopulations in tumors, which could shed light on developing new cancer diagnosis and treatment strategies based on gene dosage and epigenetic modification Citation Format: Sixue Liu, Zuyu Yang, Guanghao Li, Yanting Luo, Qiang Gong, Xin Wu, Tao Li, Zhiqian Zhang, Baocai Xing, Xiaolan Xu, Xuemei Lu. Multi-omics analysis in primary cell cultures reveals the genomic basis of phenotypic diversity within tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 208.

This paper introduces our recent experimental results of pulse tube refrigerator driven by linear compressor. The working frequency is 23-30 Hz, which is much higher than the G-M type cooler (the developed cryocooler will be called high frequency pulse tube refrigerator in this paper). To achieve a temperature below 10 K, two types of two-stage configuration, gas coupled and thermal coupled, have been designed, built and tested. At present, both types can achieve a no-load temperature below 10 K by using only one compressor. As to gas-coupled HPTR, the second stage can achieve a cooling power of 16 mW/10K when the first stage applied a 400 mW heat load at 60 K with a total input power of 400 W. As to thermal-coupled HPTR, the designed cooling power of the first stage is 10W/80K, and then the temperature of the second stage can get a temperature below 10 K with a total input power of 300 W. In the current preliminary experiment, liquid nitrogen is used to replace the first coaxial configuration as the precooling stage, and a no-load temperature 9.6 K can be achieved with a stainless steel mesh regenerator. Using Er3Ni sphere with a diameter about 50-60 micron, the simulation results show it is possible to achieve a temperature below 8 K. The configuration, the phase shifters and the regenerative materials of the developed two types of two-stage high frequency pulse tube refrigerator will be discussed, and some typical experimental results and considerations for achieving a better performance will also be presented in this paper.