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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.

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.SignificanceAcquired 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.

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.SignificanceMAPKi 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.

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.

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) T cells 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 T cell 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.

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, BRAF non-V600, and BRAF V600 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 BRAF V600MUT 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.

Objective: To investigate the prognostic value of DNA methylation of tumor suppressor genes for hepatocellular carcinoma (HCC) recurrence after liver transplantation. Methods:APC gene was selected according to The Cancer Genome Atlas dataset. Tumor tissues and clinical data of 85 HCC patients who received a liver transplantation were retrospectively enrolled and next-generation methylation sequencing was performed. Risk factors were determined using the Cox proportional-hazard-regression model. Results: The APC methylation site (chromosome 5, position 112043544) was an independent predictor of post-transplant HCC recurrence. Patients with hyper-methylated APC112043544 experienced superior recurrence-free survival (p = 0.021) and had a decreased proportion of microvascular invasion (p = 0.017). APC112043544 also predicted recurrence risk in patients beyond selection criteria. Conclusions:APC112043544 methylation may serve as a potential biomarker for post-transplant HCC recurrence. [ABSTRACT FROM AUTHOR]

Surface wave inversion based on dispersion curves of higher modes is increasingly being applied to estimate near-surface S -wave velocity structures using active or passive sources. Moreover, sensitivity analysis of dispersion curves is important for estimating the confidence of inverted results. In this study, we define a sensitivity function and a sensitivity kernel, give their analytic expressions and then propose a method to estimate the confidence of inverted results and redesign the initial structure through sensitivity analysis. Three kinds of common layered models [velocity-increasing-with-depth (regular), high-velocity layer (HVL), and low-velocity layer (LVL)] are considered. Results show that dispersion curves are more sensitive to S -wave velocity than P -wave velocity and density. The frequency band of the sensitivity function increases as the modes of dispersion curves increase, and the higher modes can penetrate additional depth. For the HVL model, the dispersion curves are particularly sensitive to those of layers above the HVL. By contrast, for the LVL model, the dispersion curves are particularly sensitive to the LVL. Aside from the analysis of sensitivity, the sensitivity functions can also be used for inversion. Thus, we perform an S -wave velocity inversion for synthetic records calculated from the structural model of Shanghai, China. Synthetic noise records are converted into the frequency–velocity domain (c-f panel) by the vector wavenumber transform method. Then, the dispersion curves of multimode are determined from the peaks of the c-f panel. The inverted structure perfectly matches the target structure except the deeper structures that is consistent with the sensitivity analysis of the determined dispersion curves. Finally, we apply the same procedure to the real case in Linyi, Shandong, China. The first three modes of dispersion curves accurately match the data. The synthetic and real examples indicate that the combined use of higher modes offers additional constraint for the velocity structure, which leads to better results of inversion than that of only the fundamental mode. In addition, the sensitivity analysis offers us a tool to estimate the confidence of the inverted structure and redesign the initial structure. [ABSTRACT FROM AUTHOR]

In the last two decades, surface-wave inversion methods such as multi-channel analysis of surface waves (MASW) have increasingly been used in engineering prospecting because of its convenience and high-efficiency. In this paper, a new multi-channel surface wave analysis method based on a Vector Wavenumber Transformation Method (VWTM) is proposed. By approximating the source, we obtain the empirical Green's function between station and the source based on the horizontal layered model, and then perform vector wavenumber transformation to obtain a frequency-phase velocity dispersion energy image for the Rayleigh wave. We first evaluate the performance of VWTM by applying it to the synthetic data, and by comparing to the theoretical dispersion curves. Comparing with typical MASW method such as the phase shift method, we find that VWTM has higher resolution and imaging quality for fundamental and higher modes in the dispersion energy image. Next, we apply the VWTM to the multi-channel transient surface wave exploration. Through comparative analysis with the phase shift method, we find that VWTM is convenient, practical and effective to extract multi-mode Rayleigh wave dispersion curves, and provide a useful alternative for the inversion of Rayleigh wave dispersion fundamental and higher-mode curves. [ABSTRACT FROM AUTHOR]

Objective: To investigate the prognostic value of DNA methylation of tumor suppressor genes for hepatocellular carcinoma (HCC) recurrence after liver transplantation. Methods: APC gene was selected according to The Cancer Genome Atlas dataset. Tumor tissues and clinical data of 85 HCC patients who received a liver transplantation were retrospectively enrolled and next-generation methylation sequencing was performed. Risk factors were determined using the Cox proportional-hazard-regression model. Results: The APC methylation site (chromosome 5, position 112043544) was an independent predictor of post-transplant HCC recurrence. Patients with hyper-methylated APC 112043544 experienced superior recurrence-free survival (p = 0.021) and had a decreased proportion of microvascular invasion (p = 0.017). APC 112043544 also predicted recurrence risk in patients beyond selection criteria. Conclusions: APC 112043544 methylation may serve as a potential biomarker for post-transplant HCC recurrence.

Development of hepatocellular carcinoma (HCC) is a dynamic process that includes a spectrum ranging from precancerous exposure to carcinogenesis and metastasis stages. In this process, numerous dysregulated genes resulted in aberrant activation or inhibition of signaling pathways. Herein, time-series gene expression profiles of dimethylnitrosamine (DEN)-induced mice with HCC covering different stages are provided. Gene expression patterns of liver tissues were detected at different time intervals [0 (negative control; NC), 15, 28, 30, and 42 weeks]. A comparison of gene expression between DEN-treated groups and NC yielded a total of 726 differentially expressed genes (DEGs), 76 of which were enriched in 10 statistically significant Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways (adjusted p value <0.05). After assessing regulation among these cascades, we found that Stat3 was a crucial transcription factor. Additionally, it was a connector in the PPI network constituting the 76 DEGs. Western blotting and immunohistochemistry suggested that the phosphorylation of Stat3 at tyrosine 705 (pStat3 Y705) was down-regulated in early stage HCC. Following, survival analysis revealed that patients with down-regulated pSTAT3 Y705 exhibited reduced overall survival rates in both the early stage and well-differentiated groups (p=0.00022 and p=0.0026, respectively). This is the first study evaluating dynamic gene expression profiles in a time-series DEN-induced mouse HCC model. Stat3 was identified as a crucial node during HCC progression, and pSTAT3 Y705 serves as a prognostic biomarker for early-stage HCC., Competing Interests: None., (AJTR Copyright © 2020.)