Timothy J. Purwin

RAC1 P29S regulates PD-L1 expression in melanoma

Whole exome sequencing of cutaneous melanoma has led to the detection of P29 mutations in RAC1 in 5–9% of samples, but the role of RAC1 P29 mutations in melanoma biology remains unclear. Using reverse phase protein array analysis to examine the changes in protein/phospho‐protein expression, we identified cyclin B1, PD‐L1, Ets‐1, and Syk as being selectively upregulated with RAC1 P29S expression and downregulated with RAC1 P29S depletion. Using the melanoma patient samples in TCGA, we found PD‐L1 expression to be significantly increased in RAC1 P29S patients compared to RAC1 WT as well as other RAC1 mutants. The finding that PD‐L1 is upregulated suggests that oncogenic RAC1 P29S may promote suppression of the antitumor immune response. This is a new insight into the biological function of RAC1 P29S mutations with potential clinical implications as PD‐L1 is a candidate biomarker for increased benefit from treatment with anti‐PD1 or anti‐PD‐L1 antibodies.

An in vivo reporter to quantitatively and temporally analyze the effects of CDK4/6 inhibitor-based therapies in melanoma

Aberrant cell-cycle progression is a hallmark feature of cancer cells. Cyclin-dependent kinases 4 and 6 (CDK4/6) drive progression through the G1 stage of the cell cycle, at least in part, by inactivating the tumor suppressor, retinoblastoma. CDK4/6 are targetable and the selective CDK4/6 inhibitor, palbociclib, was recently FDA approved for the treatment of estrogen receptor-positive, HER2-negative advanced breast cancer. In cutaneous melanoma, driver mutations in NRAS and BRAF promote CDK4/6 activation, suggesting that inhibitors such as palbociclib are likely to provide therapeutic benefit in combination with BRAF inhibitors and/or MEK inhibitors that are FDA-approved. However, the determinants of the response to CDK4/6 inhibitors alone and in combination with other targeted inhibitors are poorly defined. Furthermore, in vivo systems to quantitatively and temporally measure the efficacy of CDK4/6 inhibitors and determine the extent that CDK activity is reactivated during acquired resistance are lacking. Here, we describe the heterogeneous effects of CDK4/6 inhibitors, the expression of antiapoptotic proteins that associate with response to CDK4/6 and MEK inhibitors, and the development of a luciferase-based reporter system to determine the effects of CDK4/6 inhibitors alone and in combination with MEK inhibitors in melanoma xenografts. These findings are likely to inform on-going and future clinical trials utilizing CDK4/6 inhibitors in cutaneous melanoma. Cancer Res; 76(18); 5455-66. ©2016 AACR.

ERK-mediated phosphorylation regulates SOX10 sumoylation and targets expression in mutant BRAF melanoma

In human mutant BRAF melanoma cells, the stemness transcription factor FOXD3 is rapidly induced by inhibition of ERK1/2 signaling and mediates adaptive resistance to RAF inhibitors. However, the mechanism underlying ERK signaling control of FOXD3 expression remains unknown. Here we show that SOX10 is both necessary and sufficient for RAF inhibitor-induced expression of FOXD3 in mutant BRAF melanoma cells. SOX10 activates the transcription of FOXD3 by binding to a regulatory element in FOXD3 promoter. Phosphorylation of SOX10 by ERK inhibits its transcription activity toward multiple target genes by interfering with the sumoylation of SOX10 at K55, which is essential for its transcription activity. Finally, depletion of SOX10 sensitizes mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo. Thus, our work discovers a novel phosphorylation-dependent regulatory mechanism of SOX10 transcription activity and completes an ERK1/2/SOX10/FOXD3/ERBB3 axis that mediates adaptive resistance to RAF inhibitors in mutant BRAF melanoma cells.In BRAF mutant melanoma, inhibition of ERK1/2 induces FOXD3 and mediates RAF inhibitor resistance. Here, the authors show that ERK1/2 mediated phosphorylation regulates sumoylation of SOX10 which activates FOXD3, and depletion of SOX10 sensitises BRAF mutant melanoma cells to RAF inhibitors.

Co-targeting HGF-cMET signaling with MEK inhibitors in metastatic uveal melanoma

Patients with metastatic uveal melanoma usually die within 1 year of diagnosis, emphasizing an urgent need to develop new treatment strategies. The liver is the most common site of metastasis. Mitogen-activated protein kinase kinase (MEK) inhibitors improve survival in V600 BRAF–mutated cutaneous melanoma patients but have limited efficacy in patients with uveal melanoma. Our previous work showed that hepatocyte growth factor (HGF) signaling elicits resistance to MEK inhibitors in metastatic uveal melanoma. In this study, we demonstrate that expression of two BH3-only family proteins, Bim-EL and Bmf, contributes to HGF-mediated resistance to MEK inhibitors. Targeting HGF/cMET signaling with LY2875358, a neutralizing and internalizing anti-cMET bivalent antibody, and LY2801653, a dual cMET/RON inhibitor, overcomes resistance to trametinib provided by exogenous HGF and by conditioned medium from primary hepatic stellate cells. We further determined that activation of PI3Kα/γ/δ isoforms mediates the resistance to MEK inhibitors by HGF. Combination of LY2801653 with trametinib decreases AKT phosphorylation and promotes proapoptotic PARP cleavage in metastatic uveal melanoma explants. Together, our data support the notion that selectively blocking cMET signaling or PI3K isoforms in metastatic uveal melanoma may break the intrinsic resistance to MEK inhibitors provided by factors from stromal cells in the liver. Mol Cancer Ther; 16(3); 516–28. ©2017 AACR.

MIG6 Is MEK Regulated and Affects EGF-Induced Migration in Mutant NRAS Melanoma

Activating mutations in NRAS are frequent driver events in cutaneous melanoma. NRAS is a GTP-binding protein, whose most well-characterized downstream effector is RAF leading to activation of MEK-ERK1/2 signaling. While there are no FDA-approved targeted therapies for melanoma patients with a primary mutation in NRAS, one form of targeted therapy that has been explored is MEK inhibition. In clinical trials, MEK inhibitors have shown disappointing efficacy in mutant NRAS patients, the reasons for which are unclear. To explore the effects of MEK inhibitors in mutant NRAS melanoma, we utilized a high-throughput reverse-phase protein array (RPPA) platform to identify signaling alterations. RPPA analysis of phospho-proteomic changes in mutant NRAS melanoma in response to trametinib indicated a compensatory increase in AKT signaling and decreased expression of mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR/ERBB receptors. MIG6 expression did not alter the growth or survival properties of mutant NRAS melanoma cells. Rather, we identified a role for MIG6 as a negative regulator of EGF-induced signaling and cell migration and invasion. In MEK inhibited cells, further depletion of MIG6 increased migration and invasion, whereas MIG6 expression decreased these properties. Therefore, a decrease in MIG6 may promote the migration and invasiveness of MEK-inhibited mutant NRAS melanoma especially in response to EGF stimulation.

In Vivo E2F Reporting Reveals Efficacious Schedules of MEK1/2–CDK4/6 Targeting and mTOR–S6 Resistance Mechanisms

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance.Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568-81. ©2018 AACR.See related commentary by Sullivan, p. 532See related article by Romano et al., p. 556This article is highlighted in the In This Issue feature, p. 517.

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.

Abstract 5852: FGF2 confers resistance to novel BET inhibitors in metastatic uveal melanoma

Uveal melanoma (UM) is the most common intraocular malignancy in adults and frequently metastasizes to the liver. Metastatic UM responds poorly to chemotherapy, immunotherapy and MAPK pathway inhibitors; hence, there is an urgent unmet need for new treatment strategies. UM has a low mutational burden and progression is associated with abberrant transcriptional programs. Inhibition of epigenetic modulators involved in transcriptional regulation such as the bromodomain and extraterminal domain (BET) proteins, may be feasible for the treatment of UM. Pre-clinically, JQ1, a first generation BET inhibitor, has been shown to suppress UM growth. Additionally, a multi-site clinical trial is ongoing testing a novel BET inhibitor, PLX51107 (Plexxikon Inc) in metastatic UM patients. Since growth factors in the tumor microenvironment potentially contribute to intrinsic resistance and poor responses to therapies, we determined effects of growth factors on the efficacy of BET inhibitors in metastatic UM cells. We show that FGF2 provides resistance of UM cells to the growth inhibitory effects of BET inhibitors, JQ1, PLX51107 and the third generation inhibitor, PLX72853. FGF2 rescued BET inhibitor-induced apoptosis and cell cycle arrest, which was associated with reversal of the decreased expression of cell cycle regulators (cyclin D1, phospho-RB) and increased expression of pro-apoptotic markers (cleaved PARP, Bim). Importantly, FGF receptor (FGFR) inhibitors reversed FGF2-induced BET inhibitor resistance. As UM predominantly metastasizes to the liver, we investigated FGF2 production by hepatic stellate cells. We show that hepatic stellate cells secrete FGF2 and stellate cell conditioned medium protects metastatic UM cells against the growth inhibitory effects of BET inhibitors. Interestingly, BET inhibitor treatment of UM cells elevated FGFR protein expression and this finding is also shown in UM cell xenograft tumors and in patient tumor samples following PLX51107 treatment. These results indicate that the FGF2/FGFR pathway is activated to promote resistance to BET inhibition via the secretion of FGF2 in the tumor stroma and adaptive upregulation of FGFR expression in UM. In conclusion, our studies suggest that co-targeting of FGFR signaling and BET proteins may be required to maximize the responses of metastatic UM to BET inhibitors. Citation Format: Vivian Chua, Timothy Purwin, Connie Liao, Andrew Aplin. FGF2 confers resistance to novel BET inhibitors in metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5852.

ErbB3 Targeting Enhances the Effects of MEK Inhibitor in Wild-Type BRAF/NRAS Melanoma

MEK-ERK1/2 signaling is elevated in melanomas that are wild-type for both BRAF and NRAS (WT/WT), but patients are insensitive to MEK inhibitors. Stromal-derived growth factors may mediate resistance to targeted inhibitors, and optimizing the use of targeted inhibitors for patients with WT/WT melanoma is a clinical unmet need. Here, we studied adaptive responses to MEK inhibition in WT/WT cutaneous melanoma. The Cancer Genome Atlas data set and tumor microarray studies of WT/WT melanomas showed that high levels of neuregulin-1 (NRG1) were associated with stromal content and ErbB3 signaling. Of growth factors implicated in resistance to targeted inhibitors, NRG1 was effective at mediating resistance to MEK inhibitors in patient-derived WT/WT melanoma cells. Furthermore, ErbB3/ErbB2 signaling was adaptively upregulated following MEK inhibition. Patient-derived cancer-associated fibroblast studies demonstrated that stromal-derived NRG1 activated ErbB3/ErbB2 signaling and enhanced resistance to a MEK inhibitor. ErbB3- and ErbB2-neutralizing antibodies blocked the protective effects of NRG1 in vitro and cooperated with the MEK inhibitor to delay tumor growth in both cell line and patient-derived xenograft models. These results highlight tumor microenvironment regulation of targeted inhibitor resistance in WT/WT melanoma and provide a rationale for combining MEK inhibitors with anti-ErbB3/ErbB2 antibodies in patients with WT/WT cutaneous melanoma, for whom there are no effective targeted therapy options.Significance: This work suggests a mechanism by which NRG1 regulates the sensitivity of WT NRAS/BRAF melanomas to MEK inhibitors and provides a rationale for combining MEK inhibitors with anti-ErbB2/ErbB3 antibodies in these tumors. Cancer Res; 78(19); 5680-93. ©2018 AACR.

Abstract 5199: An in vivo reporter to quantitatively and temporally analyze the effects of CDK4/6 inhibitor-based therapies in melanoma

Aberrant cell cycle progression is a hallmark feature of cancer cells. Cyclin-dependent kinase 4 and 6 (CDK4/6) drive progression through the G1 stage of the cell cycle, at least in part, by inactivating the tumor suppressor, retinoblastoma (RB). CDK4/6 are targetable and the selective CDK4/6 inhibitor, palbociclib (IBRANCE/PD-0332991), was recently FDA approved for the treatment of estrogen receptor-positive, HER2-negative advanced breast cancer. In cutaneous melanoma, driver mutations in NRAS and BRAF signal to promote CDK4/6 activation suggesting that inhibitors such as palbociclib are likely to provide therapeutic benefit most likely in combination with BRAF and/or MEK inhibitors that are FDA-approved. However, the determinants of the response to CDK4/6 inhibitors alone and in combination with other targeted inhibitors are poorly defined. Furthermore, in vivo systems to quantitatively and temporally measure the efficacy of CDK4/6 inhibitors and determine the extent that CDK activity is reactivated during acquired resistance are lacking. Here, we describe the heterogeneous effects of CDK4/6 inhibitors, the expression of anti-apoptotic proteins that associate with response to CDK4/6 and MEK inhibitors, and the development of a luciferase-based reporter system to determine the effects of CDK4/6 inhibitors alone and in combination with MEK inhibitors in melanoma xenografts. These findings are likely to inform on-going and future clinical trials utilizing CDK4/6 inhibitors in cutaneous melanoma. Citation Format: Jessica L. Teh, Timothy Purwin, Evan J. Greenawalt, Inna Chervoneva, Allison Goldberg, Michael E. Davies, Andrew Aplin. An in vivo reporter to quantitatively and temporally analyze the effects of CDK4/6 inhibitor-based therapies in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5199.