Branka Mijatov

BRAF Inhibitor Resistance Mechanisms in Metastatic Melanoma: Spectrum and Clinical Impact

Purpose: Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma. Experimental Design: Fifty-nine BRAFV600-mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes. Results: Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive. Conclusions: Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required. Clin Cancer Res; 20(7); 1965–77. ©2014 AACR.

Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma.

Acquired resistance to BRAF inhibitors often involves MAPK re‐activation, yet the MEK inhibitor trametinib showed minimal clinical activity in melanoma patients that had progressed on BRAF‐inhibitor therapy. Selective ERK inhibitors have been proposed as alternative salvage therapies. We show that ERK inhibition is more potent than MEK inhibition at suppressing MAPK activity and inhibiting the proliferation of multiple BRAF inhibitor resistant melanoma cell models. Nevertheless, melanoma cells often failed to undergo apoptosis in response to ERK inhibition, because the relief of ERK‐dependent negative feedback activated RAS and PI3K signalling. Consequently, the combination of ERK and PI3K/mTOR inhibition was effective at promoting cell death in all resistant melanoma cell models, and was substantially more potent than the MEK/PI3K/mTOR inhibitor combination. Our data indicate that a broader targeting strategy concurrently inhibiting ERK, rather than MEK, and PI3K/mTOR may circumvent BRAF inhibitor resistance, and should be considered during the clinical development of ERK inhibitors.

The BH3-mimetic ABT-737 sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors but does not reverse acquired resistance

Although the introduction of selective v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors has been a major advance in treatment of metastatic melanoma, approximately 50% of patients have limited responses including stabilization of disease or no response at all. This study aims to identify a novel means of overcoming resistance of melanoma to killing by BRAF inhibitors. We examined the influence of the BH3-mimetic ABT-737 on induction of apoptosis by the selective BRAF inhibitor PLX4720 in melanoma cells with or without BRAF V600E mutation. Included were cell lines established from four patients before and during treatment with selective BRAF inhibitors and 3D spheroids derived from these cell lines. Cell lines with no or low sensitivity to PLX4720 underwent synergistic increases and increased rates of apoptosis when combined with ABT-737. This degree of synergism was not seen in cell lines without BRAF V600E mutations. Apoptosis was mediated through the mitochondrial pathway and was due in part to upregulation of Bim as shown by inhibition of apoptosis following small interfering RNA knockdown of Bim. Similar effects were seen in cell lines established from patients prior to treatment but not in lines from patients clinically resistant to the selective BRAF inhibitors and in 3D spheroids derived from these cell lines. These results suggest that combination of selective BRAF inhibitors with ABT-737 or the related orally available compound ABT-263 may increase the degree and rate of responses in previously untreated patients with V600E melanoma but not in those with acquired resistance to these agents.

Control of NF‐kB activity in human melanoma by bromodomain and extra‐terminal protein inhibitor I‐BET151

The transcription factor NF‐kappaB (NF‐kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF‐kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti‐apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra‐terminal (BET) protein inhibitor I‐BET151 to inhibit NF‐kB in melanoma cells. We show that I‐BET151 is a potent, selective inhibitor of a number of NF‐kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL‐6 and IL‐8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF‐kB and that I‐BET151 caused transcriptional downregulation of the NF‐kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF‐kB may have a key pathogenic role.

Mutant B-RAF-Mcl-1 survival signaling depends on the STAT3 transcription factor

Approximately 50% of melanomas depend on mutant B-RAF for proliferation, metastasis and survival. The inhibition of oncogenic B-RAF with highly targeted compounds has produced remarkable albeit short-lived clinical responses in B-RAF mutant melanoma patients. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may provide new strategies for managing melanoma. Oncogenic B-RAFV600E is known to promote the stabilizing phosphorylation of the anti-apoptotic protein Mcl-1, implicated in melanoma survival and chemoresistance. We now show that B-RAFV600E signaling also induces the transcription of Mcl-1 in melanocytes and melanoma. We demonstrate that activation of STAT3 serine-727 and tyrosine-705 phosphorylations is promoted by B-RAFV600E activity and that the Mcl-1 promoter is dependent on a STAT consensus-site for B-RAF-mediated activation. Consequently, suppression of STAT3 activity disrupted B-RAFV600E-mediated induction of Mcl-1 and reduced melanoma cell survival. We propose that STAT3 has a central role in the survival and contributes to chemoresistance of B-RAFV600E melanoma.

Oncogenic B-RAF V600E Signaling Induces the T-Box3 Transcriptional Repressor to Repress E-Cadherin and Enhance Melanoma Cell Invasion

Approximately 50% of melanomas require oncogenic B-RAFV600E signaling for proliferation, survival and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, albeit short term, clinical responses. Re-activation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole genome expression analyses to reveal that oncogenic B-RAFV600E regulates genes associated with epithelial-mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3 and E-cadherin. We show that B-RAFV600E induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues, promoting cell motility but is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway plays a critical role in promoting the metastasis of B-RAF mutant melanomas.

Histone Deacetylases (HDACs) as Mediators of Resistance to Apoptosis in Melanoma and as Targets for Combination Therapy with Selective BRAF Inhibitors

HDACs are viewed as enzymes used by cancer cells to inhibit tumor suppressor mechanisms. In particular, we discuss their role as suppressors of apoptosis in melanoma cells and as mediators of resistance to selective BRAF inhibitors. Synergistic increases in apoptosis are seen when pan-HDAC inhibitors are combined with selective BRAF inhibitors. Moreover, cell lines from patients with acquired resistance to Vemurafenib undergo PLX4720 induced apoptosis when combined with pan-HDAC inhibitors. The mechanisms of upregulation of HDACs and the mechanisms involved in HDACi reversal of resistance to apoptosis are as yet poorly understood.

Identification of a single amino acid residue which is critical for the interaction between HSV-1 inner tegument proteins pUL36 and pUL37.

The herpes simplex virus type 1 (HSV-1) structural tegument proteins pUL36 and pUL37 are essential for secondary envelopment during the egress of viral particles. For this study, scanning alanine mutagenesis of HSV-1 pUL37, in combination with yeast two-hybrid, identified pUL37 residue D631 as a major determinant for binding of pUL36. Further analysis of the binding of this pUL37 mutant to pUL36 by coimmunoprecipitation assay confirmed the role of pUL37 D631 in mediating binding of pUL36. A trans-complementation assay using pUL37 deletion virus FRΔUL37 was then carried out, where pUL37 wild type or D631A were provided in trans. For pUL37 D631A, a significant reduction in virus titer was observed compared to that seen when pUL37 wild type was present. The results presented here underline the crucial role of the pUL36/pUL37 interaction in replication of HSV-1 and indicate a critical role for pUL37 D631 in mediating this interaction.

Overcoming Resistance of Melanoma to Immunotherapy with Monoclonal Antibodies Against Checkpoints Inhibitors

The introduction of monoclonal antibodies against checkpoint inhibitors on T cells such as Ipilimumab and anti PD1 has been a major advance in the treatment of metastatic melanoma. Nevertheless, the majority of patients does not receive benefit from this treatment approach. The reasons for this are likely to reflect conditioning of the microenvironment of the tumour by properties of the tumour cells that either prevent infiltration by T cells or which modulate their function. Treatments that combine monoclonal antibodies with agents that inhibit melanoma cell production of suppressive cytokines and which favor infiltration of T cells into the tumour are the focus of ongoing studies.