Kevin J. Basile

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.

TWIST1 is an ERK1/2 effector that promotes invasion and regulates MMP-1 expression in human melanoma cells

Tumor cells often use developmental processes to progress toward advanced disease. The E-box transcription factor TWIST1 is essential to epithelial-mesenchymal transition (EMT) and cell migration in the developing neural crest. In melanoma, which derives from the neural crest cell lineage, enhanced TWIST1 expression has been linked to worse clinical prognosis. However, mechanisms underlying TWIST1 expression and whether aberrant TWIST1 levels promote steps in melanoma progression remain unknown. Here, we report that elevated TWIST1 mRNA/protein expression is dependent on extracellular signal-regulated kinase (ERK)1/2 signaling, which is hyperactive in the majority of melanomas. We show that TWIST1 protein levels are especially high in melanoma cell lines generated from invasive, premetastatic stage tumors. Furthermore, TWIST1 expression is required and sufficient to promote invasion through Matrigel and spheroid outgrowth in three-dimensional dermal-mimetic conditions. Alterations to spheroid outgrowth were not as a result of altered cell death, cell-cycle profile, or paradigm EMT protein changes. Importantly, we identify matrix metalloproteinase-1 (MMP-1) as a novel downstream target of TWIST1. We have determined that TWIST1 acts, in a dose-dependent manner, as a mediator between hyperactive ERK1/2 signaling and regulation of MMP-1 transcription. Together, these studies mechanistically show a previously unrecognized interplay between ERK1/2, TWIST1, and MMP-1 that is likely significant in the progression of melanoma toward metastasis.

Adaptive upregulation of FOXD3 and resistance to PLX4032/4720-induced cell death in mutant B-RAF melanoma cells.

Melanoma cells driven by mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) are highly resistant to chemotherapeutic treatments. Recent phase 1 results with PLX4032/RG7204/vemurafenib, which selectively inhibits B-RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field. Nearly all patients in the phase 1–3 studies saw at least some response and the overall response rates ranged from 48 and 81%. However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time. These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032. It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond. We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is upregulated following inhibition of B-RAF–MEK signaling in mutant B-RAF melanoma cells. Here, we show that upregulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death. Small interfering RNA-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines. Additionally, upregulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death. Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment. Together, these data indicate that upregulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance.

Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAFV600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAFV600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAFV600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAFV600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAFV600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.

In Vivo MAPK Reporting Reveals the Heterogeneity in Tumoral Selection of Resistance to RAF Inhibitors

Activation of the ERK1/2 mitogen-activated protein kinases (MAPK) confers resistance to the RAF inhibitors vemurafenib and dabrafenib in mutant BRAF-driven melanomas. Methods to understand how resistance develops are important to optimize the clinical use of RAF inhibitors in patients. Here, we report the development of a novel ERK1/2 reporter system that provides a noninvasive, quantitative, and temporal analysis of RAF inhibitor efficacy in vivo. Use of this system revealed heterogeneity in the level of ERK1/2 reactivation associated with acquired resistance to RAF inhibition. We identified several distinct novel and known molecular changes in resistant tumors emerging from treatment-naïve cell populations including BRAF V600E variants and HRAS mutation, both of which were required and sufficient for ERK1/2 reactivation and drug resistance. Our work offers an advance in understanding RAF inhibitor resistance and the heterogeneity in resistance mechanisms, which emerge from a malignant cell population.

Beneficial effects of RAF inhibitor in mutant BRAF splice variant-expressing melanoma.

Resistance to RAF inhibitors such as vemurafenib and dabrafenib is a major clinical problem in the treatment of melanoma. Patients with mutant BRAF melanoma that progress on RAF inhibitors have limited treatment options, and drug removal from resistant tumors may elicit multiple effects. A frequent mechanism of resistance to RAF inhibitors is caused by expression of mutant BRAF splice variants. RAF inhibitor–resistant cell lines, generated in vivo, were tested as to whether or not mutant BRAF splice variants confer a fitness advantage in the presence of RAF inhibitor. Critically, cells expressing distinct mutant BRAF splice variants grow more efficiently in vitro and in vivo in the presence of the vemurafenib analog, PLX4720, compared with in the absence of inhibitor. PLX4720-treated BRAF splice variant–expressing cells exhibited levels of phospho-extracellular signal–regulated kinase (ERK)1/2 comparable to untreated parental cells. In addition, a reduction in phospho-ERK1/2 levels following treatment with the MEK inhibitor, trametinib (GSK1120212) phenocopied the fitness benefit provided by PLX4720. These data indicate that mutant BRAF splice variant–expressing melanoma cells are benefited by defined concentrations of RAF inhibitors. Implications: This study provides evidence that RAF inhibitor–resistant melanoma cells benefit from continued therapy. Mol Cancer Res; 12(5); 795–802. ©2014 AACR.

Resistance to chemotherapy: short-term drug tolerance and stem cell-like subpopulations.

Personalized medicine in cancer treatment has been a major goal for decades. Recently, the development of several therapies that specifically target key genetic alterations in different malignancies has dramatically improved patient outcome and brought the goal of personalized medicine closer to practicality. Despite the improved specificity of these treatment options, resistance to targeted therapy is common and remains a major obstacle to long-term management of a patients disease. Often patient relapse is a result of the positive selection of cells with certain genetic alterations that result in a bypass of the therapeutic intervention. Once this occurs, patient relapse is inevitable and further treatment options are limited. The time to relapse is often quite rapid indicating that cancer cells may be primed for adapting to cytotoxic stimuli. Recently, it has been suggested that small subpopulations of cells allow resistance to occur more rapidly. It is thought that these cells are capable of surviving strong apoptotic stimuli until more permanent mechanisms of long-term resistance are developed. In order to decrease the rate of patient relapse, more studies are required in order to identify these subpopulations of cells, understand the mechanisms underlying their drug tolerance, and develop strategies to prevent them from evading treatment.

Chapter Ten - Resistance to Chemotherapy: Short-Term Drug Tolerance and Stem Cell-Like Subpopulations

Personalized medicine in cancer treatment has been a major goal for decades. Recently, the development of several therapies that specifically target key genetic alterations in different malignancies has dramatically improved patient outcome and brought the goal of personalized medicine closer to practicality. Despite the improved specificity of these treatment options, resistance to targeted therapy is common and remains a major obstacle to long-term management of a patients disease. Often patient relapse is a result of the positive selection of cells with certain genetic alterations that result in a bypass of the therapeutic intervention. Once this occurs, patient relapse is inevitable and further treatment options are limited. The time to relapse is often quite rapid indicating that cancer cells may be primed for adapting to cytotoxic stimuli. Recently, it has been suggested that small subpopulations of cells allow resistance to occur more rapidly. It is thought that these cells are capable of surviving strong apoptotic stimuli until more permanent mechanisms of long-term resistance are developed. In order to decrease the rate of patient relapse, more studies are required in order to identify these subpopulations of cells, understand the mechanisms underlying their drug tolerance, and develop strategies to prevent them from evading treatment.

Abstract 3928: In vivo ERK1/2 reporting demonstrates the association and timing of ERK1/2 pathway reactivation and acquired resistance to RAF inhibitors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Patients with V600 mutant BRAF positive metastatic melanoma are now treated with the RAF inhibitor, vemurafenib (Zelboraf), as a first line therapy. Although most patients treated with vemurafenib have tumor shrinkage, other patients exhibit disease progression. Furthermore, most patients who initially respond to the drug eventually develop acquired resistance to vemurafenib. Disease progression while on vemurafenib frequently correlates with ERK1/2 pathway reactivation but previous studies have been unable to precisely monitor the temporal changes in ERK1/2 activity. Here, we establish a cell-based ERK1/2 reporter system that provides quantitative analysis of RAF inhibitor action in mutant BRAF melanoma cells in vitro and in vivo. This system was utilized to show that the RAF inhibitor, PLX4720, effectively targets the ERK1/2 pathway in vivo and that ERK1/2 reactivation is associated with tumor re-growth. Levels of ERK1/2 reactivation varied substantially between tumors and were associated with several different molecular changes. These data present a new system from monitoring in vivo efficacy of ERK1/2 pathway inhibitors and identifying mechanisms of in vivo acquired resistance. Citation Format: Kevin J. Basile, Ethan V. Abel, Neda Dadpey, Andrew E. Aplin. In vivo ERK1/2 reporting demonstrates the association and timing of ERK1/2 pathway reactivation and acquired resistance to RAF inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2013-3928

Resistance to Chemotherapy

Personalized medicine in cancer treatment has been a major goal for decades. Recently, the development of several therapies that specifically target key genetic alterations in different malignancies has dramatically improved patient outcome and brought the goal of personalized medicine closer to practicality. Despite the improved specificity of these treatment options, resistance to targeted therapy is common and remains a major obstacle to long-term management of a patients disease. Often patient relapse is a result of the positive selection of cells with certain genetic alterations that result in a bypass of the therapeutic intervention. Once this occurs, patient relapse is inevitable and further treatment options are limited. The time to relapse is often quite rapid indicating that cancer cells may be primed for adapting to cytotoxic stimuli. Recently, it has been suggested that small subpopulations of cells allow resistance to occur more rapidly. It is thought that these cells are capable of surviving strong apoptotic stimuli until more permanent mechanisms of long-term resistance are developed. In order to decrease the rate of patient relapse, more studies are required in order to identify these subpopulations of cells, understand the mechanisms underlying their drug tolerance, and develop strategies to prevent them from evading treatment.