Pilot Study of ONCOS-102 and Pembrolizumab: Remodeling of the Tumor Microenvironment and Clinical Outcomes in Anti-PD-1-Resistant Advanced Melanoma.

PURPOSE: Intratumoral oncolytic virotherapy may overcome anti-PD(L)-1 resistance by triggering pro-inflammatory remodeling of the tumor microenvironment. This pilot study investigated ONCOS-102 (oncolytic adenovirus expressing GM-CSF) plus anti-programmed cell death protein 1 (PD)-1 therapy in anti-PD-1-resistant melanoma. PATIENTS AND METHODS: Patients with advanced melanoma progressing after prior PD-1 blockade received intratumoral ONCOS-102 either as priming with 3 doses (3 × 1011 viral particles) during Week 1 [Part 1 (sequential treatment)] or as 4-dose priming and 8 booster doses every 3 weeks [Part 2 (combination treatment)]. From Week 3, all patients received pembrolizumab every 3 weeks (≤8 doses). The primary endpoint was safety. Objective response rate (ORR), progression-free survival, and immunologic activation in repeat biopsies were also investigated. RESULTS: In 21 patients (Part 1, n = 9; Part 2, n = 12) ONCOS-102 plus pembrolizumab was well tolerated: most adverse events (AE) were mild/moderate in severity. Pyrexia (43%), chills (43%), and nausea (28%) were the most common ONCOS-102-related AEs. There were no dose-limiting toxicities. ORR was 35% [response evaluation in solid tumors (RECIST) 1.1, irRECIST]. Reduction in size of ≥1 non-injected lesions observed in 53% patients indicated a systemic effect. In injected tumors, persistent immune-related gene expression and T-cell infiltration were associated with clinical benefit. Viral persistence and efficacy in injected and non-injected lesions without additional toxicity supported Part 2 dosing regimen in future studies. CONCLUSIONS: ONCOS-102 plus pembrolizumab was well tolerated and led to objective responses in patients with anti-PD-1-resistant advanced melanoma. ONCOS-102 promoted T-cell infiltration, particularly cytotoxic CD8+ T cells, which persisted at Week 9, driving clinical benefit. Further investigation of ONCOS-102 plus PD-1 blockade is warranted. See related commentary by Levi and Boland, p. 3.

B6-Dct-H2BGFP bitransgenic mice: A standardized mouse model for in vivo characterization of melanocyte development and stem cell differentiation.

Melanocyte stem cells (McSCs) are key components of the hair follicle (HF) stem cell system that regenerate differentiated melanocytes during successive HF cycles. To facilitate continued research on melanocyte development and differentiation and McSCs, we backcrossed inducible Dct-H2BGFP mice into the C57BL/6J background (B6-Dct-H2BGFP). We compared the expression pattern of B6-Dct-H2BGFP to that of Dct-H2BGFP mice on a mixed genetic background reported previously. To characterize B6-Dct-H2BGFP mice, we confirmed not only the expression of GFP in all melanocyte lineage cells, but also doxycycline regulation of GFP expression. Furthermore, ex vivo culture of the McSC subsets isolated by fluorescence-activated cell sorting (FACS) showed the propensity of bulge/CD34+ McSCs to differentiate with expression of non-melanocytic, neural crest lineage markers including glia (Gfap and CNPase, 73 ± 1% and 77 ± 2%, respectively), neurons (Tuj1 26 ± 5%), and smooth muscle (α-Sma, 31 ± 9%). In contrast, CD34-/secondary hair germ (SHG) McSCs differentiated into pigmented melanocytes, with higher expression of melanogenic markers Tyr (71 ± 1%), Tyrp1 (68 ± 4%), and Mitf (75 ± 7%). These results establish the utility of B6-Dct-H2BGFP bitransgenic mice for future in vivo studies of melanocytes requiring a defined genetic background.

Cellular Phenotypic Plasticity of Cutaneous Melanoma: A Complex Puzzle.

Wenzina et al. (2020) explore the potential role of E-cadherin (CDH1) as a marker for invasive behavior in melanoma. The authors show that CDH1 expression is modulated by p38 signaling, and that manipulation of this pathway can impede endothelial disruption and lung dissemination in vivo and in vitro. The downstream markers PODXL and DEL of the invasive phenotype are associated with a poor prognosis.

A Pilot Study of Sirolimus in Subjects with Cowden Syndrome or Other Syndromes Characterized by Germline Mutations in PTEN.

LESSONS LEARNED: This is the first human interventional study in patients with Cowden syndrome that is driven by inactivation of germline PTEN gene.Single-agent sirolimus, a mTOR inhibitor, suppressed mTOR signaling in surrogate human tissues without significant toxicity. BACKGROUND: Cowden syndrome is characterized by inactivating germline PTEN mutations, which can lead to activation of the PI3K-Akt-mTOR pathway. METHODS: Adult subjects with germline PTEN mutation who met international diagnostic criteria for Cowden syndrome and who had Eastern Cooperative Oncology Group (ECOG) performance status 0-2 and adequate organ function were enrolled. Subjects were treated with a 56-day course of daily oral sirolimus. In addition to symptom assessment and physical examination, dermatologic, endoscopic, neurologic (cerebellar), and radiographic assessments were conducted. Inhibition of the mTOR pathway in benign skin and gastrointestinal (GI) lesion was assessed by immunohistochemistry. RESULTS: A total of 18 patients and 16 families were enrolled. PTEN mutations were located at exons 1-8. Regression of skin and GI lesions was observed by dermoscopy or endoscopy. Neurological evaluation showed improvement in cerebellar function score at 1 month. Immunohistochemistry (IHC) analysis in skin and GI benign lesions showed a decrease in the ratio of phosphorylated (p)S6 to total S6 in response to sirolimus. Ratios of pS6K to total S6 at days 14 and 56 were significantly lower than at baseline (p = .0026, p = .00391, respectively). A 56-day course of sirolimus was well tolerated. CONCLUSION: A 56-day course of sirolimus was well tolerated in subjects with Cowden syndrome and was associated with some evidence of improvement in symptoms, skin and GI lesions, cerebellar function, and decreased mTOR signaling.

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.

Characterization of a new, inducible transgenic mouse model with GFP expression in melanocytes and their precursors.

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties.

Melanocytic Nevi and the Genetic and Epigenetic Control of Oncogene-Induced Senescence.

Melanocytic nevi represent benign clonal proliferations of the melanocytes in the skin that usually remain stable in size and behavior or disappear during life. Infrequently, melanocytic nevi undergo malignant transformation to melanoma. Understanding molecular and cellular mechanisms underlying oncogene-induced senescence should help identify pathways underlying melanoma development, leading to the development of new strategies for melanoma prevention and early detection.

Polycomb group proteins--epigenetic repressors with emerging roles in melanocytes and melanoma.

Melanocytes undergo rapid and significant changes in their gene expression programs at regular intervals during development and the hair follicle cycle. In melanoma, the gene expression pattern found in normal melanocytes is disrupted. These gene expression patterns are regulated in part by post-translational histone modifications catalyzed by Polycomb group (PcG) proteins, which play a major role in many developmental processes and are often altered in cancer. In this review, we discuss the role of the PcG proteins in stem cell and cancer biology, in general, as well as in melanocyte development and melanomagenesis. Highlights include the discussion of newly identified treatments that target the activity of PcG proteins as well as new developments in the understanding of the role that these proteins play in melanocyte biology.

High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients.

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV-type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho-S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV-related XP nevi and melanomas were different from nevi and melanomas in the general population.

Epigenetic cancer prevention mechanisms in skin cancer.

Epigenetics is an important emerging area for study of mechanisms of cancer prevention. In recent years, it has been realized that cancer prevention agents, derived from natural dietary sources, impact cancer cell survival by modulating epigenetic processes. In the present manuscript, we review key epigenetic regulatory mechanisms and examine the impact of sulforaphane and green tea polyphenols on these processes. We also discuss available information on the epigenetics in the context of skin cancer. These studies indicate that diet-derived chemopreventive agents modulate DNA methylation status and histone modification via multiple processes and point to additional areas for study of epigenetic mechanisms in skin cancer.

Hearing dysfunction in heterozygous Mitf(Mi-wh) /+ mice, a model for Waardenburg syndrome type 2 and Tietz syndrome.

The human deafness-pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia-White (Mitf(Mi-wh) /+) mice were studied and hearing function of these mice characterized. Mitf(Mi-wh) /+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf(Mi-wh) /+ embryos have fewer melanoblasts during embryonic development than their wild-type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf(Mi-wh) /+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness-pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.

EZH2-dependent suppression of a cellular senescence phenotype in melanoma cells by inhibition of p21/CDKN1A expression.

Polycomb group (PcG) proteins such as Enhancer of zeste homolog 2 (EZH2) are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. Targets of PcG include cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. Senescence is a characteristic of melanocytic nevi, benign melanocytic proliferations that can be precursors of malignant melanoma. In this study, we report that EZH2, which we find absent in melanocytic nevi but expressed in many or most metastatic melanoma cells, functionally suppresses the senescent state in human melanoma cells. EZH2 depletion in melanoma cells inhibits cell proliferation, restores features of a cellular senescence phenotype, and inhibits growth of melanoma xenografts in vivo. p21/CDKN1A is activated upon EZH2 knockdown in a p53-independent manner and contributes substantially to cell cycle arrest and induction of a senescence phenotype. EZH2 depletion removes histone deacetylase 1 (HDAC1) from the CDKN1A transcriptional start site and downstream region, enhancing histone 3 acetylation globally and at CDKN1A. This results in recruitment of RNA polymerase II, leading to p21/CDKN1A activation. Depletion of EZH2 synergistically activates p21/CDKN1A expression in combination with the HDAC inhibitor trichostatin A. Since melanomas often retain wild-type p53 function activating p21, our findings describe a novel mechanism whereby EZH2 activation during tumor progression represses p21, leading to suppression of cellular senescence and enhanced tumorigenicity.

Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.

Future advances in melanoma research.

The future of melanoma research is promising. Specific mechanisms leading to oncogenic transformation in melanoma development have been identified, and are likely to produce new targets for melanoma therapy. Also, advances in melanoma research will result from melanoma investigators co-opting approaches used to study other malignancies in which progress has been made more rapidly. Systematic roadblocks limiting advances in melanoma research relative to other malignancies are being addressed in a formal manner. The public and public officials are increasingly becoming aware of the need for more dedicated efforts to address the challenges of research on this malignancy.

SKI knockdown inhibits human melanoma tumor growth in vivo.

The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Evidence of ultraviolet type mutations in xeroderma pigmentosum melanomas.

To look for a direct role of ultraviolet radiation (UV) exposure in cutaneous melanoma induction, we studied xeroderma pigmentosum (XP) patients who have defective DNA repair resulting in a 1000-fold increase in melanoma risk. These XP melanomas have the same anatomic distribution as melanomas in the general population. We analyzed laser capture microdissection samples of skin melanomas from XP patients studied at the National Institutes of Health. The tumor suppressor gene PTEN was sequenced and analyzed for UV-induced mutations. Samples from 59 melanomas (47 melanomas in situ and 12 invasive melanomas) from 8 XP patients showed mutations in the PTEN tumor suppressor gene in 56% of the melanomas. Further, 91% of the melanomas with mutations had 1 to 4 UV type base substitution mutations (occurring at adjacent pyrimidines) (P < 0.0001 compared to random mutations). We found a high frequency of amino-acid-altering mutations in the melanomas and demonstrated that these mutations impaired PTEN function; UV damage plays a direct role in induction of mutations and in inactivation of the PTEN gene in XP melanomas including in situ, the earliest stage of melanoma. This gene is known to be a key regulator of carcinogenesis and therefore these data provide solid mechanistic support for UV protection for prevention of melanoma.

Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.

Microphthalmia-associated transcription factor (Mitf) is essential for melanocyte development and function and regulates anti-apoptotic Bcl2 expression. We hypothesized that cellular deficiency of Mitf can influence melanocyte survival in response to ultraviolet (UV) radiation. Primary melanocyte cultures were prepared from neonatal wild-type mice and congenic animals heterozygous for Mitf mutations Mitf (mi-vga9/+) and Mitf(Mi-wh/+) and exposed to UV irradiation. Wild-type melanocytes were more resistant to UV-induced apoptosis than melanocytes partially deficient in Mitf activity, as determined by relative levels of intracellular melanin and relative activation of Mitf target genes Tyr, Tyrp1, Dct, and Cdk2. Comparative experiments with wild-type cells and congenic albino melanocytes demonstrated that these differences are not due to differences in melanin content, implicating Mitf as a primary determinant of UV-dependent melanocyte survival. Mitf activity correlated directly with resistance to UV-induced apoptosis in melanocytes. Mitf was important not only for regulating the expression of anti-apoptotic Bcl-2 following UV irradiation, but also the expression of the pro-apoptotic BH3-only Bad protein and activation of the extrinsic apoptotic pathway. Hence, Mitf is a multifaceted regulator of UV-induced apoptosis in melanocytes.