Few effective therapeutic options exist after progression on immune checkpoint blockade (ICB) for melanoma. Here, we utilized a platform incorporating transcriptomic profiling, high-throughput drug screening, and murine models to demonstrate the preclinical efficacy of several combinatorial regimens against ICB-resistant melanoma. Transcriptomic analysis of ICB-resistant melanomas demonstrated activation of several targetable pathways. High-throughput drug screening targeting these pathways identified several effective combinations in ICB-resistant patient-derived xenograft models. The combination of cobimetinib and regorafenib (termed Cobi+Reg) emerged as a particularly promising regimen, with efficacy against distinct molecular melanoma subtypes and after progression on ICB in immunocompetent models. Transcriptomic and spatial analysis of Cobi+Reg-treated tumors demonstrated upregulation of antigen presentation machinery, with concomitantly increased activated T cell infiltration. Combining Cobi+Reg with ICB was superior to either modality in vivo. This analytical platform exploits the biology of ICB-resistant melanoma to identify therapeutic vulnerabilities, resulting in the identification of drug combinations that form the basis for rational clinical trial design in the setting of advanced melanoma resistant to ICB.

Supplementary Figure Legends 1-4, Methods from Temporal Dissection of Tumorigenesis in Primary Cancers

Supplementary Figure 4 from Temporal Dissection of Tumorigenesis in Primary Cancers

Supplementary Figure 3 from Temporal Dissection of Tumorigenesis in Primary Cancers

Supplementary Figure 2 from Temporal Dissection of Tumorigenesis in Primary Cancers

Supplementary Table 1 from Temporal Dissection of Tumorigenesis in Primary Cancers

Therapy of BRAF -mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF -mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS - and NF1 -mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

Significance Transcription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

Overview Skin carcinogenesis from solar UV exposure is initiated by UV photoproducts formed in the DNA of precursor cells that give rise to squamous cell carcinomas (SCC), basal cell carcinomas (BCC), and cutaneous malignant melanomas (CMM). SCC and BCC are caused by UVB but CMM by reactive oxygen generated through UVA absorption in melanin. The UVB photoproducts consist of dimers between adjacent pyrimidines that are repaired by nucleotide excision repair (NER). Two branches of NER differ in mechanisms of damage recognition: in global genome repair (GGR) damage is recognized in nontranscribed DNA by the DDB2 and XPC proteins; in transcription‐coupled repair (TCR) damage is recognized by arrest of the transcribing RNA polymerase at damaged sites. The T = C photoproduct is the predominant mutagenic product which, if unrepaired, gives rise to C to T or CC to TT mutations through replication by the low fidelity polymerase Pol H. TCR reduces the mutation frequency in regions of transcription. Deficiencies in GGR in the human disease xeroderma pigmentosum cause earlier onset and increased rates of skin cancer. Deficiencies in TCR in the human disease Cockayne syndrome are associated with photosensitivity, developmental and neurological disorders, but no cancer has been reported.

AbstractMelanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G2–M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma.Significance:This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light.

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma sub-type and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.

Background Mitotic rate is a strong, independent prognostic factor in patients with melanoma. However, incorporating it into the melanoma staging system has proved challenging. Methods The prognostic impact of mitotic rate was assessed in a melanoma cohort comprising 5050 patients from 2 geographically distinct populations. Computer‐generated cut points for mitotic rate were constructed to determine its impact on melanoma‐associated survival using Kaplan‐Meier and multivariate regression analyses. The impact of mitotic rate also was assessed in randomly split training and validation sets. Results Mitotic rate had a nonlinear impact on survival, as evidenced by unequally spaced cut points. An index incorporating these cut points that was constructed from one population produced significantly more accurate predictions of survival in the other population than using the entire scale of mitotic rate. An index constructed from the combined cohort was found to be independently predictive of survival, with an impact comparable to that of ulceration. Optimal high‐versus‐low cut points for mitotic rate were generated separately for each T category (<2 mitoses/mm 2 vs ≥2 mitoses/mm 2 for T1 melanoma, <4 mitoses/mm 2 vs ≥4 mitoses/mm 2 for T2 melanoma, <6 mitoses/mm 2 vs ≥6/mitoses/mm 2 for T3 melanoma, and <7 mitoses/mm 2 vs ≥7 mitoses/mm 2 for T4 melanoma). Using Kaplan‐Meier analysis, elevated mitotic rate was found to have an impact on survival comparable to that of ulceration within each T category. Application of the index for mitotic rate that was constructed from the training data set demonstrated an independent impact in the validation data set, with a significance similar to that of ulceration. Conclusions The results of the current study demonstrated the comparable prognostic impact of mitotic rate and ulceration, providing support for its reincorporation into the T category.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.

The U.S. Fish and Wildlife Service listed Anaxyrus canorus, the Yosemite toad, as federally threatened in 2014 based upon reported population declines and vulnerability to global-change factors. A. canorus lives only in California’s central Sierra Nevada at medium to sub-alpine elevations. Lands throughout its range are protected from development, but climate and other global-change factors potentially can limit populations. A. canorus reproduces in ultra-shallow wetlands that typically hydrate seasonally via melting of the winter snowpack. Lesser snowpacks in drier years can render wetland water volumes and hydroperiods insufficient to allow for successful breeding and reproduction. Additionally, breeding and embryogenesis occur very soon after wetlands thaw when overnight temperatures can be below freezing. Diseases, such as chytridiomycosis, which recently decimated regional populations of ranid species, also might cause declines of A. canorus populations. However, reported studies focused on whether climate interacts with any pathogens to affect fitness in A. canorus have been scarce. We investigated effects of these factors on A. canorus near Tioga Pass from 1996 to 2001. We found breeding subpopulations were distributed widely but inconsistently among potentially suitable wetlands and frequently consisted of small numbers of adults. We occasionally observed small but not alarming numbers of dead adults at breeding sites. In contrast, embryo mortality often was notably high, with the majority of embryos dead in some egg masses while mortality among coincidental Pseudacris regilla (Pacific treefrog) embryos in deeper water was lower. After sampling and experimentation, we concluded that freezing killed A. canorus embryos, especially near the tops of egg masses, which enabled Saprolegnia diclina (a water mold [Oomycota]) to infect and then spread through egg masses and kill more embryos, often in conjunction with predatory flatworms (Turbellaria spp.). We also concluded exposure to ultraviolet-B radiation did not play a role. Based upon our assessments of daily minimum temperatures recorded around snow-off during years before and after our field study, the freezing potential we observed at field sites during embryogenesis might have been commonplace beyond the years of our field study. However, interactions among snow quantity, the timing of snow-off, and coincidental air temperatures that determine such freezing potential make projections of future conditions highly uncertain, despite overall warming trends. Our results describe important effects from ongoing threats to the fitness and abundance of A. canorus via reduced reproduction success and demonstrate how climate conditions can exacerbate effects from pathogens to threaten the persistence of amphibian populations.

Melanoma to Vitiligo: The Melanocyte in Biology & Medicine generated and amplified a growing understanding of the scientific basis of and clinical and pathologic relationship between melanoma, a by-product of uncontrolled growth of melanocytes, and vitiligo, a disorder of undesirable death of melanocytes. This meeting, held October 17–22, 2018, at Salishan Resort on the Oregon Coast, incorporated the full spectrum of pigment cell biology and chemistry, as well as the biological underpinnings of clinical disease.

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis.

Melanoma patients with additional positive lymph nodes in the completion lymph node dissection (CLND) following a positive sentinel lymph node (SLN) biopsy would have a poorer prognosis than patients with no additional positive lymph nodes. We hypothesize that the progression of disease from the SLN to the non-SLN compartment is orderly and is associated with the worsening of the disease status. Thus, the SLN and non-SLN compartments are biologically different in that cancer cells, in general, arrive in the SLN compartment before spreading to the non-SLN compartment. To validate this concept, we used a large cohort of melanoma patients from our prospective SLN database in an academic tertiary medical center. Adult cutaneous melanoma patients (n = 291) undergoing CLND after a positive SLN biopsy from 1994 to 2009 were analyzed. Comparison of 5-year disease-free survival and 5-year overall survival between positive (n = 66) and negative (n = 225) CLND groups was made. The 5-year disease-free survival rates were 55% (95% CI 49–62%) for patients with no additional LN on CLND versus 14% (95% CI 8–26%) in patients with positive LN on CLND (p < 0.0001, log-rank test). The median disease-free survival time was 7.4 years with negative CLND (95% CI 4.4–15+ years) and 1.2 years with positive CLND (95% CI 1.0–1.8 years). The 5-year overall survival rates were 67% (95% CI 61–74%) for negative CLND versus 38% (95% CI 28–52%) for positive CLND (p < 0.0001, log-rank test). The median overall survival time was 12.1 years for negative CLND (95% CI 9.3–15+ years) and 2.5 years for positive CLND (95% CI 2.2–5.7 years). This study shows that CLND status is a significant prognostic factor for patients with positive SLNs undergoing CLND. Also, it suggests an orderly progression of metastasis from the SLN to the non-SLN compartment. Thus, the SLN in the regional nodal basin draining the primary melanoma may serve as an important gateway for metastasis to the non-SLN compartment and beyond to the systemic sites.

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.