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SnapshotDx Quiz: November 2021

  • Meiqi Luo
    Affiliations
    Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
    Search for articles by this author
  • Emily Y. Chu
    Correspondence
    Correspondence: Emily Y. Chu, Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, 2 Maloney Building, 3600 Spruce Street, Philadelphia, Pennsylvania 19104, USA.
    Affiliations
    Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
    Search for articles by this author

      What is your Diagnosis?

      Figure thumbnail gr1
      Figure 1
      Image courtesy of Emily Y. Chu, Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
      Editorial note: Welcome to the Journal of Investigative Dermatology (JID) SnapshotDx Quiz. In this monthly online-only quiz, the first question (“What is your diagnosis?”) relates to the clinical image shown, while additional questions concern the findings reported in a JID article by
      • Guhan S.M.
      • Shaughnessy M.
      • Rajadurai A.
      • Taylor M.
      • Kumar R.
      • Ji Z.
      • et al.
      The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
      (https://doi.org/10.1016/j.jid.2020.12.035).
      Detailed answers and a list of relevant references are available following the Quiz Questions below.

      Quiz Questions

      • 1.
        What is your diagnosis?
        • a.
          Spitz nevus
        • b.
          Melanoma in situ
        • c.
          Pigmented basal cell carcinoma
        • d.
          Pigmented squamous cell carcinoma in situ
        • e.
          Dysplastic nevus
      • 2.
        According to
        • Guhan S.M.
        • Shaughnessy M.
        • Rajadurai A.
        • Taylor M.
        • Kumar R.
        • Ji Z.
        • et al.
        The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
        , the sensitivity of BRAF/NRAS/NF1–wild-type (BNFwt) melanomas to CDK9 inhibition could be attributed to their dependencies on a transcriptional network. Which of the following findings best supports this statement?
        • a.
          Genetic ontology reveals that genes that are depleted in all BRAF/NRAS/NF1-mutant (BNFmut) and BNFwt cell lines are associated with transcriptional factors.
        • b.
          Pol-II-carboxy-terminal domain is consistently abrogated by NVP-2 treatment.
        • c.
          Two-dimensional and three-dimensional cell cultures showed significantly more cell death in BNFwt cell lines than in BNFmut cell lines on CDK9 inhibition.
        • d.
          Multiple E2F factors exhibit greater decrement in BNFwt than in BNFmut in response to CDK9 inhibition.
        • e.
          High levels of E2F1, E2F2, E2F3, and E2F6 expression were associated with worsened survival.
      • 3.
        Targeted therapies exist for melanomas with which of the following genotypes?
        • a.
          BRAF mutated
        • b.
          NRAS mutated
        • c.
          NF1 mutated
        • d.
          Triple wild type
        • e.
          GNAQ mutated
      See following pages for detailed answers

      Detailed Answers

      • 1.
        What is your diagnosis?
      • CORRECT ANSWER: b. Melanoma in situ
      • This photomicrograph shows a melanoma in situ, with confluent growth of nested and single atypical melanocytes along the dermal‒epidermal junction and pagetoid spread. Within this melanoma in situ, there is variability in the size of the nests, and the lesion demonstrates marked asymmetry.
        • Discussion of incorrect answers:
        • a.
          Spitz nevus: Spitz nevi are benign melanocytic skin lesions with epithelioid and/or spindled melanocytes and are most commonly seen in children and young adults. Melanocytes in Spitz nevi are found in nests, which show peripheral clefting, often with a raining-down appearance. Pagetoid spread may be seen in the center of the lesions. Kamino bodies are frequently observed. Spitz nevi typically appear symmetrical and organized, which distinguish them from the melanoma in situ shown in this figure.
        • c.
          Pigmented basal cell carcinoma: On histopathology, basal cell carcinomas typically display islands or nests of basaloid cells with peripheral palisading at the border. Approximately 2–5% of lesions in basal cell carcinomas are pigmented, which can mimic malignant melanomas. Functional melanocytes can be found scattered within these tumor islands.
        • d.
          Pigmented squamous cell carcinoma in situ: On histopathology, pigmented squamous cell carcinoma in situ shows full-thickness atypia of keratinocytes with associated melanin pigmentation. Pagetoid spread may be seen in squamous cell carcinoma in situ, but nested growth of atypical cells as seen in this melanoma in situ is not a typical feature.
        • e.
          Dysplastic nevus: Dysplastic nevi are characterized by architectural disorder of melanocytic nests in the epidermis, manifesting as a fusion of melanocytic nests in adjacent rete pegs or as bridging. In addition, there may be crowded growth of nested and some single melanocytes along the dermal–epidermal junction. In addition, random cytological atypia and stromal response are characteristic findings in dysplastic nevi. The confluent growth of severely atypical melanocytes seen in this photomicrograph is not characteristic of a dysplastic nevus.
      • 2.
        According to
        • Guhan S.M.
        • Shaughnessy M.
        • Rajadurai A.
        • Taylor M.
        • Kumar R.
        • Ji Z.
        • et al.
        The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
        , the sensitivity of BRAF/NRAS/NF1–wild-type (BNFwt) melanomas to CDK9 inhibition could be attributed to their dependencies on a transcriptional network. Which of the following findings best supports this statement?
      • CORRECT ANSWER: d. Multiple E2F factors exhibit greater decrement in BNFwt than in BNFmut in response to CDK9 inhibition.
      • To determine the molecular pathways that distinguish BNFwt from BRAF/NRAS/NF1-mutant (BNFmut) tumor cell lines,
        • Guhan S.M.
        • Shaughnessy M.
        • Rajadurai A.
        • Taylor M.
        • Kumar R.
        • Ji Z.
        • et al.
        The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
        identified 2,029 genes that were differentially expressed between BNFwt and BNFmut after NVP-2 treatment. Genes that were more significantly depleted in BNFwt than in BNFmut were subjected to transcription factor (TF) target enrichment and network analyses with chromatin immunoprecipitation–X enrichment analysis 3 (ChEA3), which reveal a significant association with the E2F network. E2F family members are TFs that play a critical role in regulating gene expression during cell proliferation. On NVP-2 treatment, several E2F family members showed a significantly greater decrement in BNFwt, confirming the results from ChEA3 analysis and TF interactome. Furthermore, E2F1 and E2F2 are expressed at a significantly higher level in BNFwt than in BNFmut melanomas without CDK9 inhibition. Following these findings,
        • Guhan S.M.
        • Shaughnessy M.
        • Rajadurai A.
        • Taylor M.
        • Kumar R.
        • Ji Z.
        • et al.
        The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
        drew a direct link between transcriptional network and CDK9 inhibition by showing a significant correlation between CDK9 expression and E2F1/2 expression levels. Taken together, these results suggest that BNFwt melanomas can be distinguished from BNFmut melanomas by differential expression of the E2F network, and their dependencies on this transcription network may contribute to their vulnerability to CKD9 inhibition.
        • Discussion of incorrect answers:
        • a.
          Genetic ontology reveals that genes that are depleted in all BRAF/NRAS/NF1-mutant (BNFmut) and BNFwt cell lines are associated with transcriptional factors: Using RNA sequencing,
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          identified 2,659 genes that were depleted ≥2-fold in all the eight tumor cell lines (including both BNFwt and BNFmut) after NVP-2 treatment. The most significant genetic ontology annotations for molecular functions were TFs. These findings suggest that the changes seen in CDK9 inhibition could be explained by a depletion of genes that are enriched for TFs. However, depletion of these genes is not limited to BNFwt but occurs in both BNFwt and BNFmut. Although the findings raised the suspicion that BNFwt is more susceptible to the depletion of these genes, they do not directly explain why BNFwt has a more notable response to CDK9 inhibition.
        • b.
          Pol-II-carboxy-terminal domain is consistently abrogated by NVP-2 treatment: Figure 3a of
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          shows the level of CDK9 and its phosphorylated serine-2 (pSer2) carboxy-terminal domain (CTD) in BNFmut and BNFwt cell lines after treatment with either TS-032 or NVP-2, both of which are CDK9 inhibitors. Both CDK9 and Pol-II-CTD (pSer2), a known target of CDK9, are abrogated to different extents by NVP-2 and TS-032 across all samples. These findings illustrate the effects of TS-035 and NVP-2 on BNFmut and BNFwt cell lines but do not explain why BNFwt are more sensitive to these effects.
        • c.
          Two-dimensional and three-dimensional cell cultures showed significantly more cell death in BNFwt cell lines than in BNFmut cell lines on CDK9 inhibition: In Figure 2 of
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          , BNFwt cell lines were shown to have a significantly higher number of dead cells with TS-032 or NVP-2 treatment than BNFmut and primary cells. These results show the sensitivity of BNFwt to CDK9 inhibition but do not explain the underlying mechanism of this sensitivity.
        • e.
          High levels of E2F1, E2F2, E2F3, and E2F6 expression were associated with worsened survival: Using data among the Cancer Genome Atlas skin cutaneous melanoma cohort,
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          found that high expression levels of E2F1, E3F3, E2F3, E2F6 and low expression level of E2F5 are associated with worse survival. These TFs belong to the E2F family and play critical roles in regulating gene expression during cell proliferation. This finding suggests that the E2F family might be associated with a more aggressive oncogenic network, and pharmacological depletion of these oncogenic factors could be achieved through the inhibition of their transcription. However, this finding does not implicate a dependency of BNFwt melanomas on the E2F transcriptional network.
      • 3.
        Targeted therapies exist for melanomas with which of the following genotypes?
      • CORRECT ANSWER: a. BRAF mutated
      • BRAF mutations occur in approximately 50% of melanomas, with BRAF V600E being the most commonly seen genotype (https://www.cancer.gov/tcga). These mutations lead to the activation of BRAF kinase and its downstream kinases MAPK/extracellular signal–regulated kinase (ERK) kinase (MEK) and ERK, resulting in uncontrolled cell proliferation. Therefore, inhibitors that target BRAF are included as the first-line treatment for these tumors. For example, combination treatment with BRAF inhibitors such as dabrafenib and MEK inhibitors such as trametinib has shown improved patient outcomes in BRAF V600‒mutated melanomas (
        • Eroglu Z.
        • Ribas A.
        Combination therapy with BRAF and MEK inhibitors for melanoma: latest evidence and place in therapy.
        ). In addition, BRAF-mutant tumors are susceptible to immune checkpoint PD-1 inhibitors such as ipilimumab because focal amplifications of PD-L1 gene are observed at a high frequency among BRAF-mutant melanomas (
        Cancer Genome Atlas Network
        Genomic classification of cutaneous melanoma.
        ). However, the overall survival rate of advanced BRAF melanomas is still low at around 50% and 28% even with the use of checkpoint inhibitors and BRAF inhibitors, respectively (
        • Long G.V.
        • Eroglu Z.
        • Infante J.
        • Patel S.
        • Daud A.
        • Johnson D.B.
        • et al.
        Long-term outcomes in patients with BRAF V600–mutant metastatic melanoma who received dabrafenib combined with trametinib.
        ;
        • Wolchok J.D.
        • Chiarion-Sileni V.
        • Gonzalez R.
        • Rutkowski P.
        • Grob J.J.
        • Cowey C.L.
        • et al.
        Overall survival with combined nivolumab and ipilimumab in advanced melanoma [published correction appears in N Engl J Med 2018;379:2185].
        ).
        • Discussion of incorrect answers:
        • b.
          NRAS mutated: NRAS mutations occur in around 20–30% melanomas, most of which harbor the Q61R genotype (https://www.cancer.gov/tcga). Mutations in NRAS, a member of the small GTPase RAS family, result in constitutive activation of downstream pathways, including the RAS‒RAF‒MAPK and phosphoinositide 3-kinase (PI3K)‒protein kinase B (Akt) pathways, leading to dysregulation in cell proliferation (
          • Hodis E.
          • Watson I.R.
          • Kryukov G.V.
          • Arold S.T.
          • Imielinski M.
          • Theurillat J.P.
          • et al.
          A landscape of driver mutations in melanoma.
          ). Clinically, NRAS-mutant melanomas are more aggressive than other subtypes, with thicker lesions, elevated mitotic activity, and higher rates of lymph node metastasis (
          • Devitt B.
          • Liu W.
          • Salemi R.
          • Wolfe R.
          • Kelly J.
          • Tzen C.Y.
          • et al.
          Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma.
          ). Although MEK inhibitors and immune-based therapies have been employed in the treatment of NRAS-mutant melanomas, managing these tumors remains challenging owing to the lack of NRAS-targeted therapies and rapid development of drug resistance (
          • Muñoz-Couselo E.
          • Adelantado E.Z.
          • Ortiz C.
          • García J.S.
          • Perez-Garcia J.
          NRAS-mutant melanoma: current challenges and future prospect.
          ).
        • c.
          NF1 mutated: NF1 mutations are present in 10–15% of melanomas, 30% of which co-occur with BRAF/NRAS mutations (
          Cancer Genome Atlas Network
          Genomic classification of cutaneous melanoma.
          ). NF1 gene encodes neurofibromin, a RAS GTPase‒activating protein that downregulates MAPK activity. Mutations in NF1 result in a loss of its tumor-suppressing function and lead to dysregulated cellular proliferation (
          • Cirenajwis H.
          • Lauss M.
          • Ekedahl H.
          • Törngren T.
          • Kvist A.
          • Saal L.H.
          • et al.
          NF1-mutated melanoma tumors harbor distinct clinical and biological characteristics.
          ). Similar to NRAS-mutant melanomas, whereas MEK inhibitors may be employed in the treatment for this genomic subtype, a specific targeted therapy does not exist for NF1-mutated melanoma (
          • Py C.
          • Christinat Y.
          • Kreutzfeldt M.
          • Mckee T.A.
          • Dietrich P.Y.
          • Tsantoulis P.
          Response of NF1-mutated melanoma to an MEK inhibitor.
          ).
        • d.
          Triple wild type: About 20% of melanomas lack oncogenic variants in BRAF, NRAS, or NF1 but are associated with other less common mutated genes such as GNAQ, GNA11, and KIT (
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          ). These tumors harbor far fewer mutations than BRAF-, NRAS-, and NF1-mutant tumors, resulting in the lack of druggable targets and poor response to checkpoint inhibitors (
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          ). According to
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          , CDK9 suppression was found to confer a differential response between triple wild-type (BNFwt) melanomas and BNFmut melanomas, which could be attributed to transcriptional dependencies in BNFwt tumors (
          • Guhan S.M.
          • Shaughnessy M.
          • Rajadurai A.
          • Taylor M.
          • Kumar R.
          • Ji Z.
          • et al.
          The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
          ). Their findings suggest that transcriptional inhibition could be a potential treatment for these therapeutically orphaned tumors.
        • e.
          GNAQ mutated: GNAQ-mutant melanoma is a genetic subtype of the triple wild-type melanoma. GNAQ encodes Gαq, which is a member of the G-protein-coupled receptor family proteins and has diverse functions in cell survival and signaling. Mutations of GNAQ are most often found in uveal melanoma (32%), blue nevi (32%), and cutaneous melanomas (1.4%) (
          • Shoushtari A.N.
          • Carvajal R.D.
          GNAQ and GNA11 mutations in uveal melanoma.
          ). These activating mutations in GNAQ lead to constitutive phosphorylation of the bound guanine nucleotide, increasing downstream signaling (
          • Van Raamsdonk C.D.
          • Bezrookove V.
          • Green G.
          • Bauer J.
          • Gaugler L.
          • O’Brien J.M.
          • et al.
          Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.
          ). There are diverse downstream targets for GNAQ-mutant melanomas such as the MEK/MAPK and the PI3K/Akt pathways, and combined therapy of MEK + PI3K inhibition has shown promising preclinical results. However, a drug that specifically targets GNAQ does not exist (
          • Larribère L.
          • Utikal J.
          Update on GNA alterations in cancer: implications for uveal melanoma treatment.
          ).

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        Combination therapy with BRAF and MEK inhibitors for melanoma: latest evidence and place in therapy.
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        The molecular context of vulnerability for CDK9 suppression in triple wild-type melanoma.
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        Update on GNA alterations in cancer: implications for uveal melanoma treatment.
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      Linked Article

      • The Molecular Context of Vulnerability for CDK9 Suppression in Triple Wild-Type Melanoma
        Journal of Investigative DermatologyVol. 141Issue 8
        • Preview
          Approximately half of melanoma tumors lack a druggable target and are unresponsive to current targeted therapeutics. One proposed approach for treating these therapeutically orphaned tumors is by targeting transcriptional dependencies (oncogene starvation), whereby survival factors are depleted through inhibition of transcriptional regulators. A drug screen identified a CDK9 inhibitor (SNS-032) to have therapeutic selectivity against wild-type (wt) BRAFwt/NRASwt melanomas compared with BRAFmut/NRASmut mutated melanomas.
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