Curtis H. Kugel

Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors.

sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

SHOC2 and CRAF Mediate ERK1/2 Reactivation in Mutant NRAS-mediated Resistance to RAF Inhibitor

Background: Reactivation of ERK1/2 frequently underlies acquired resistance to RAF inhibitors. Results: NRAS mutations are acquired during resistance to RAF inhibitors and promote CRAF and SHOC2-modulated ERK1/2 pathway re-activation. Conclusion: NRAS mutations in mutant BRAF cells alter RAF isoform and SHOC2 usage in the presence of RAF inhibitor. Significance: These studies delineate mechanisms mediating RAF inhibitor resistance in mutant BRAF cells. ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAFV600E melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAFV600E/NRASQ61K co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAFV600 cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAFV600E/NRASQ61K cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAFV600E/NRASQ61K cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.

Adaptive resistance to RAF inhibitors in melanoma

The discovery of activating mutations in BRAF at high frequency in cutaneous melanoma opened the door to new treatment options, which have resulted in significantly better patient outcomes. Treatments such as the FDA‐approved RAF inhibitor vemurafenib and the more recently approved dabrafenib and trametinib combination therapy are designed to target the ERK1/2 pathway. Initial success in targeting this pathway is evidenced by the high percentage of melanoma patients who undergo tumor remission. However, the beneficial effects of these targeted therapies are usually short‐lived due to the development of resistance, which leads to disease progression. As a result, studies have focused on the acquired forms of resistance that develop following continued exposure to therapy. Conversely, far fewer studies have investigated the adaptive forms of resistance, which activate rapidly, promote cell survival, and may underlie the development of acquired resistance by providing melanoma cells the time to develop additional mutations. We provide a detailed review of the known mechanisms of adaptive resistance in melanoma and relate them to similar responses to targeted therapies in other tumor types.

Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

ERBB3/HER3 expression and signaling are upregulated in mutant BRAF melanoma as an adaptive, prosurvival response to FDA-approved RAF inhibitors. Because compensatory ERBB3 signaling counteracts the effects of RAF inhibitors, cotargeting ERBB3 may increase the efficacy of RAF inhibitors in mutant BRAF models of melanoma. Here, we corroborate this concept by showing that the ERBB3 function-blocking monoclonal antibody huHER3-8 can inhibit neuregulin-1 activation of ERBB3 and downstream signaling in RAF-inhibited melanoma cells. Targeting mutant BRAF in combination with huHER3-8 decreased cell proliferation and increased cell death in vitro, and decreased tumor burden in vivo, compared with targeting either mutant BRAF or ERBB3 alone. Furthermore, the likelihood of a durable tumor response in vivo was increased when huHER3-8 was combined with RAF inhibitor PLX4720. Together, these results offer a preclinical proof of concept for the application of ERBB3-neutralizing antibodies to enhance the efficacy of RAF inhibitors in melanoma to delay or prevent tumor regrowth. As ERBB3 is often upregulated in response to other kinase-targeted therapeutics, these findings may have implications for other cancers as well.

Age Correlates with Response to Anti-PD1, Reflecting Age-Related Differences in Intratumoral Effector and Regulatory T-Cell Populations

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1. Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies. Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice. Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR. See related commentary by Pawelec, p. 5193

Inhibition of Age-Related Therapy Resistance in Melanoma by Rosiglitazone-Mediated Induction of Klotho

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals. Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor–sensitive and BRAF inhibitor–resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181–90. ©2017 AACR.

Response and Resistance to Paradox-Breaking BRAF Inhibitor in Melanomas In Vivo and Ex Vivo

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K–mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenib-resistant BRAF splice variant–expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. Mol Cancer Ther; 17(1); 84–95. ©2017 AACR.

When metastasis ‘Spns’ out of control: Coverage of ‘Genome-wide in vivo screen identifies novel host regulators of metastatic colonization’

Metastasis is the major cause of death from cancer in patients. Invasive tumor cells that leave the primary tumor must undergo a strenuous journey including extravasation, survival in the blood, intravasation into the distal site, and proliferation in a new microenvironment to form metastatic colonies. The tumor cells that are successful in forming colonies have dodged multiple perils including evading immune defenses and oxidative stress in the circulatory system. A small number of cells which leave the tumor and circulate through the blood reach the lung, and an even smaller number of tumor cells survive in the lung microenvironment, and proliferate to form metastatic foci (Fukuhara et al., 2012). Disseminated tumor cells may become dormant and take several years to ultimately grow out and form overt metastases (Massague & Obenauf, 2016). This dormancy is maintained in three ways: by cellular dormancy, where the cells themselves remain in a slowcycling state; by angiogenic dormancy, where micrometastatic populations do not have the nutrient supply to overcome their dormant state; and finally, by immunemediated dormancy, where the immune system kills some tumor cells, keeping the micrometastatic populations in check. The role of immunemediated tumor dormancy has been highlighted by clinical evidence in patients receiving kidney transplants from donors who had previously had melanoma, but who were declared to be tumorfree. Recipients of donor organs went on to develop melanoma in the transplanted kidneys (Mendoza et al., 2012). It is thought that the immunosuppressant drugs taken by these organ recipients allowed for the rapid outgrowth of the melanoma metastases, thus highlighting the importance of the immune system in maintaining tumor dormancy. Using an elegant in vivo screen, where 810 mutant mouse cell lines were screened, van der Weyden et al. have further confirmed the critical role of the host immune microenvironment in the establishment of metastases. Van der Weyden et al. identified 23 genes, which regulate metastatic colonization of the lung. Of these genes, 19 are genes previously unknown to modulate host control of metastatic colonization. While genes associated with multiple cellular processes were identified, the majority had important roles in the immune regulation of metastatic outgrowth. In this study, pulmonary metastatic colonization was found to be suppressed by eight genes (Irf1, Rnf10, Pik3cg, Dph6, Slc9a3r2, Irf7, Abhd17a), two of which are members of the interferon regulatory family (Irf1 and Irf7), which is important for immune function. While interferons have previously been implicated in the regulation of metastasis, very little is known about the microenvironmental function of the Rnf10, Dph6, Slc9a3r2, and Abhd17a genes in suppressing metastasis. Another 11 genes previously unknown to regulate metastatic colonization were also identified (Grsf1, BC017643, Fam175b, Bach2, Ncf2, Arhgef1, Fbxo7, Tbc1d22a, Lrig1, Cyba, Spns2). Of these genes, the authors chose Spns2 to explore in greater depth, as the manipulation of Spns2 had a profound impact on metastatic burden. Spns2, the sphingosine1phosphate transporter spinster homologue2, is a cell surface transporter on endothelial cells which allows for the secretion of sphingosine1phosphate (S1P) into the blood and lymph (Nagahashi et al., 2013). Previous studies suggest that Spns2 is largely responsible for lymphatic concentrations of S1P, but plays less of a role in blood concentrations of S1P (Senft & Ronai, 2016). Secretion of S1P into blood and lymph creates an S1P gradient, which promotes the egress of newly formed T cells from the thymus and B cells from the bone marrow (Strauss & Thomas, 2010). S1P is a sphingolipid which signals through extracellular Gcoupled protein receptors (S1PR15), such as S1PR1 on T cells and B cells and S1PR5 on natural killer (NK) cells. S1P is important in vascular signaling, playing a role in angiogenesis and vascular permeability, as well as immune cell trafficking. Van der Weyden et al. found S1P to be increased in the lungs and decreased in the blood of Spns2tm1a/tm1a mice; however, there was no difference in vascular permeability.

Abstract 4913: Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation

Metastatic melanoma is highly aggressively and often therapy resistant. Signaling pathways which promote invasion of metastatic melanoma, also promote therapy resistance. Therapy resistant melanomas are characterized by high levels of the pro-invasive non-canonical Wnt molecule, Wnt5A. Previously, we described an adaptive stress response in highly invasive melanoma cells, which is characterized by a growth arrest and an increase in senescence markers, yet these cells retain the ability to invade and form colonies. These highly invasive cells do not undergo apoptosis following treatment with DNA damaging agents such as doxorubicin and are resistant to BRAFV600E targeted therapy. Recently, Lukin et al. have shown that p53 expression promotes survival of colorectal cancer cells via p21 expression and a reversible cell cycle arrest, allowing for repair of damaged DNA. Here, we show that highly invasive and therapy resistant melanoma cells express Wnt5A, p53 and p21, which regulate proliferation and slow cycling in invasive melanoma cells. The expression of p53 is promoted by Wnt5A, knock down of Wnt5A decreases p53 and p21 expression in these cells and decreases the number of cells arrested in G2/M following DNA damage. The cell cycle and apoptotic functions of p53 are highly regulated. iASPP, MDMX, and MDM2 have been shown to regulate the function of wild type p53 in melanoma cells. MDM2, an E4 ubiquitin ligase, regulates p53 by shuttling it out of the nucleus and targeting it for proteasomal degradation. We found that MDM2 expression increases in invasive melanoma cells following DNA damage, however, in these cells it is phosphorylated at serine 395. Phosphorylation of MDM2 at ser395 blocks its ability to export p53 from the nucleus, leading to increased p53 expression. Even with increased p53 expression, invasive melanoma cells undergo a cell cycle arrest following stress instead of apoptosis. The apoptotic function of p53 has been shown to be inhibited by nuclear iASPP, which is enriched in metastatic melanoma. We found that knocking down Wnt5A in invasive melanoma cells decreases the expression of iASPP. These data suggest that Wnt5A promotes MDM2 Ser395 phosphorylation and iASPP expression, blocking down regulation of p53 and its apoptotic function, while promoting cell cycle arrest and survival following stress. These data may reveal a mechanism by which highly invasive melanoma cells evade therapy to form therapy resistant sub-colonies at distant sites. Citation Format: Marie R. Webster, Amanpreet Kaur, Abibatou Ndoye, Curtis Kugel, Subhasree Basu, Alexander Valiga, Jessica Appleton, Ying-Jie Wang, Maureen Murphy, Ashani T. Weeraratna. Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2017-4913