BRAF targets in melanoma. Biological mechanisms, resistance, and drug discovery. Cancer drug discovery and development. Volume 82. Ed. Ryan J. Sullivan. Springer (2015)
Limitations of BRAF-directed therapy
Functional redundancy and compensatory activity through alternate signaling pathways might explain the emergence of resistance seen in patients treated with selective BRAF inhibitors. Intense research efforts are focused on resistance mechanisms, and several mechanisms have been identified [14–21]. To address these issues, combination of BRAF/MAPK-targeted therapy with other signal transduction inhibitors, or with conventional chemotherapy has been proposed.
Combination strategies to overcome resistance have gained traction, and the combination of dabrafenib (a BRAF inhibitor) with tremetinib (a MEK inhibitor) has been FDA-approved based on an improved progression free survival (PFS) benefit in comparison to either BRAF inhibitor alone. Specifically, median PFS was extended from under 6 months for BRAF inhibitor monotherapy to over 10 months with combination BRAF + MEK inhibition . Perhaps more impressive is the percentage of patients alive without disease progression at 1 year, increasing from 10% for BRAF inhibitor monotherapy to 40% in the setting of combined BRAF inhibition and MEK inhibition . Other strategies combining MAPK inhibition with blockade of additional signaling pathways are currently in clinical trials, however data regarding response rates and durability of response are not yet available.
Despite these advances, most patients progress within a year even with the best of these combination strategies . Nonetheless this incremental benefit in survival provides a window of opportunity to offer novel agents and combination strategies, including combinations with immunotherapy. This strategy can be used on a backbone of BRAF inhibitor monotherapy or with combined BRAF and MEK inhibition, though there are important considerations with each which will be discussed herein.
Limitations of immunotherapy
Several forms of immunotherapy are either FDA-approved or in clinical trials for the treatment of metastatic melanoma. High dose IL-2 was FDA-approved in 1998 based on its ability to produce durable responses in 6–10% of patients . However, its application has been limited to a select group of patients treated in specialized centers due to its severe and unique acute toxicity .
Another form of immunotherapy that is currently FDA-approved for melanoma involves the use of a blocking antibody against the Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) molecule on the surface of T lymphocytes. CTLA4 is an immunomodulatory molecule that functions to down-regulate an immune response . Treatment with a monoclonal antibody that blocks this interaction (Ipilimumab) relieves cytotoxic T-lymphocytes from the inhibitory effects of CTLA4, resulting in an enhanced immune response. Treatment with ipilimumab has shown an overall survival advantage in patients with advanced melanoma in a randomized, placebo controlled trial  and received approval by the FDA in 2011. In this trial, patients with previously treated advanced melanoma were randomly assigned in a 3:1:1 ratio to ipilimumab plus a gp 100 vaccine, ipilimumab alone, or gp 100 alone. A significant improvement in median overall survival for patients receiving either ipilimumab containing regimen (median 10 months) relative to patients receiving the vaccine alone (6.4 months) was shown as well as a reduction of the risk of death (ipilimumab + vaccine or ipilimumab alone vs gp 100 vaccine; HR 0.68 or 0.66, respectively). Overall survival rates for the three groups were 44, 46 and 25% at 12 months and 22, 24 and 14% at 24 months, respectively .
Other forms of immunotherapy are in clinical trials and have shown promising results. Blockade of the immune-modulatory molecule PD1 on the surface of T lymphocytes has shown significant promise in the treatment of metastatic melanoma with response rates approaching 40% in a phase II clinical trial . Interestingly, responses were also seen in other solid tumors, including renal cell carcinoma and non small cell lung cancer . Monoclonal antibodies blocking the immunosuppressive ligand PDL1 are also in clinical trials, though data regarding responses and durability are not yet mature .
Another area of great promise in immunotherapy involves the use of adoptive cell transfer, and includes the administration of autologous tumor infiltrating lymphocytes (TIL) or genetically-modified peripheral blood lymphocytes (PBL) to mediate an anti-tumor response. TIL-based approaches have been quite successful in expert hands [25–27], with response rates ranging from 30 to over 70% depending on the pre-conditioning regimen used . However this therapy is still considered experimental and to date its use is limited to expert centers given the complexity and cost of generating this individualized form of treatment. Nonetheless, strategies are under development to optimize and standardize preparation of this type of product so that its use may be more generalizable. In addition, approaches using transduction of PBL with antigen-specific T cell receptors  and chimeric antigen receptors  are also underway and have shown some promising results.
The field of immunotherapy has certainly advanced the treatment of patients with metastatic melanoma, and treatment responses are often long-lasting. Unfortunately, only a minority of patients will ultimately benefit from these treatments. Thus a critical question is whether or not we can increase the durability of responses and/ or complete response rate by the addition of BRAF-directed therapy to immunotherapy regimens.
Effects of BRAF inhibition on the tumor microenvironment and immune system
Preliminary evidence suggests that oncogenic BRAF (BRAFV600E) may contribute to immune escape in melanoma , and that blocking its activity via MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (MDAs) . We studied this extensively in the laboratory, and demonstrated that targeted inhibition of the MAPK pathway leads to up to a 100-fold increase in expression of MDAs in melanoma cell lines and fresh tumor digests (Fig. 8.1a) which is associated with significantly enhanced recognition by antigen-specific T lymphocytes (Fig. 8.1b) . This appears to be mediated through microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of melanocytes .
Importantly, BRAF-directed therapy does not appear to have deleterious effects on T lymphocytes [8, 9]. This is in contrast to MEK inhibitors, which demonstrate dose-dependent inhibition on T cell function in vitro . This has relevance when contemplating combinations of BRAF-directed therapy with immunotherapy, as combination therapy including a MEK inhibitor may potentially have deleterious effects on T cells, which may abrogate any potential synergy.
Fig. 8.1. MAPK pathway inhibition increases melanoma antigen expression. Expression of MART-1 is increased with MEK inhibition and BRAF inhibition (a), which is associated with enhanced recognition by antigen-specific T lymphocytes (b), HLA-A2 + UACC903 melanoma cells were treated as above with a MEK (?U0126) or BRAF (?PLX4720) inhibitor and cultured with CTL specific for MART1 or gp100 versus control lymphocytes (GFP-transduced) at various E:T ratios. IFN? release was measured by ELISA. (Adapted from Boni et al. )
The first evidence that BRAF inhibition could result in increased immunogenicity in patients with metastatic melanoma was presented and published by several groups in 2012, demonstrating enhanced T cell infiltrates in tumors of patients with metastatic melanoma treated with BRAF inhibitors [33, 34] (Fig. 8.2a). Since these original reports, evidence regarding the immune effects of BRAF inhibition has mounted. In addition to an increase in CD8 T cell infiltrate, treatment with BRAF inhibitors is associated with a decrease in immunosuppressive cytokines IL-6, IL-8  (Fig. 8.2b) and a decrease in vascular endothelial growth factor (VEGF)  (Fig. 8.2c). The tumor stroma appears to play a critical role, as stromal cell-mediated immunosuppression via interleukin 1 (IL-1) is induced by oncogenic BRAF and blocked with BRAF inhibitors .
Fig. 8.2. BRAF inhibition is associated with increased CD8+ T-cell infiltrate, decreased immunosuppressive cytokines and VEGF in tumors of patients with metastatic melanoma. Patients with metastatic melanoma were treated with BRAF inhibitor +/MEK inhibitor and tumor biopsies were performed before treatment and within 1–2 weeks of initiation of therapy. CD8+ T cell infiltrate was assayed via immunohistochemistry (IHC) showing a significant increase of CD8+ T cells on therapy (a), This was associated with a decrease in IL-6 and IL-8 (b), as well as a decrease in VEGF (c).
An additional piece of evidence supporting the hypothesis that T cells play an important role in response to BRAF-targeted therapy and that BRAF-directed therapy may synergize with immunotherapy comes from analysis of melanoma antigen expression and CD8+ T cell infiltrate in lesions of patients who have progressed on BRAF-directed therapy . Based on our initial data, we would expect that resistance to therapy would be associated with a decrease in melanoma antigen expression and a decrease in CD8 T cell infiltrate. We tested this by analyzing melanoma antigen expression and CD8+ T cells in lesions of patients who progressed on therapy and we found exactly what we expected (Fig. 8.3), namely reduced melanoma antigen expression and CD8+ T cell infiltrate at time of progression. Interestingly, if you treat with additional MAPK blockade you can potentially restore antigen expression and T cell infiltrate (Fig. 8.3) .
Fig. 8.3. Melanoma antigen expression and CD8+ T-cell infiltrate are decreased at time of progression and restored through MEK inhibition. Tumors were harvested pre-treatment, 10–14 days after BRAFi initiation, at time of progression and at time of treatment with combined BRAF inhibition and MEK inhibition for a patient. mRNA levels of the melanoma antigens gp100, MART-1, TYRP-1, and TYRP-2 were assayed. Immunohistochemistry (IHC) was conducted for CD8+ T cells on patient tumor samples.
Another insight into tumor—stromal—T cell interactions came with the observation that the infiltrating T cells in tumors of patients treated with BRAF inhibitors demonstrate an activated phenotype and express high levels of PD-1 (Fig. 8.4a) . The PD-1 molecule is an immunomodulatory molecule that serves to down-regulate an immune response after an initial period of activation, functioning normally to prevent autoimmunity. However another critical finding in patients treated with BRAF inhibitors is that the tumor cells themselves express high levels of PD-L1 within 2 weeks of initiation of BRAF inhibitor therapy (Fig. 8.4b) . This may represent a mechanism of resistance, and is corroborated by in vitro work demonstrating high PD-L1 expression in melanoma cell lines resistant to BRAF inhibition . Interestingly, the addition of MEK inhibition may abrogate the up-regulation of PD-L1 in these cell lines in vitro, which has significant translational implications . Taken together, these data suggest that addition of an immune checkpoint inhibitor to a regimen of BRAF inhibition may augment responses to therapy (Fig. 8.5) .
Fig. 8.4. BRAF inhibition is associated with decreased markers of T-cell cytotoxicity but increased T-cell exhaustion markers and PDL1 in tumors of patients with metastatic melanoma. Tumors were harvested and mRNA levels perforin (n = 11), Granzyme B (n = 11), TIM-3 (n = 14) and PD1 (n = 14; (a), in patients with metastatic melanoma undergoing treatment with a selective inhibitor of BRAFV600E were assayed. All patients are expressed in a box and whiskers plot. Open circles represent data points greater than 1.5 times the interquartile range. P values indicated are from a 2-tailed Student t test with a µ of 1, which represents no change in mRNA value with respect to the pretreatment value. *, P = 0.05. Immunohistochemistry (Ч 40 magnification) for PDL1 in a representative pretreatment and on-treatment biopsy (b). The dotted line = tumor–stroma interface and the inset is the isotype-specific control.
Fig. 8.5. Oncogenic BRAF contributes to immune escape through the down-regulation of melanoma-differentiation antigens and by establishing an immunosuppressive tumor microenvironment. The administration of a BRAF inhibitor promotes clinical responses along with an increased expression of melanoma-differentiation antigens by malignant cells, an increased tumor infiltration by CD8+ T cells, and a decreased production of immunosuppressive cytokines such as IL-6, IL-8 and IL-1 as well as of the angiogenic mediator vascular endothelial growth factor (VEGF). This phenotype is reverted at time of disease progression. Importantly, the expression of immunomodulatory molecules on T cells (e.g., PD1) and on tumor cells (e.g., PDL1) is also increased within 14 d of BRAF-targeted therapy initiation. Taken together, these data suggest that the therapeutic potential of BRAF-targeted agents may be significantly improved by the early blockade of immune checkpoints.
Mouse models have provided important insights into cancer development, progression, therapy, and resistance. Recent melanoma models have incorporated interactions of several signature mutations found in human melanoma, enabling the generation of a mouse that recapitulates hallmark features of the disease. To date, several studies have been published showing synergy of BRAF-directed therapy in murine models [10–12, 37] and one study has shown no synergy .
The first model demonstrating synergy was published by Koya, et al. and utilized a BRAFV600E-driven murine model of melanoma, SM1, which is syngeneic to fully immunocompetent mice. In this mouse model of BRAFV600E melanoma, Koya et al. showed improved anti-tumor activity, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells in combination with a BRAF inhibitor . However, T cell analysis also showed that BRAF inhibition did not alter the expansion, distribution or tumor accumulation of adoptively transferred T cells .
Another model demonstrating synergy between BRAF-directed therapy and immunotherapy was published by Liu, et al. In this manuscript, the authors used melanoma cells transduced with gp100 and H-2Db in a xenograft model on pmel-1 TCR transgenic mice on a C57BL/6 background and found an increase in tumor infiltrate and anti-tumor activity of adoptively transferred cells after BRAF inhibition (Fig. 8.6a) . In this model, BRAF inhibition induced T-cell infiltration that was associated with a decrease in VEGF (Fig. 8.6b). In this paper they also found that VEGF overexpression in melanoma cells abrogates T cell infiltration . This corroborates what is seen in patients treated with BRAF-directed therapy, as down regulation of intratumoral VEGF correlates with increased T-cell infiltration when melanoma patients are treated with a BRAF inhibitor .
Fig. 8.6. PLX4720 increases infiltration of adoptively transferred T cells only in tumors containing BRAFV600E. B6 nude mice (5 mice/group) bearing BRAFV600E A375/H-2Db/gp 100 and BRAFT WT C918/H-2Db/gp 100 tumors were treated with OFL-expressing pmel-1 T cells, along with gp100 peptide-pulsed dendritic cells, by intravenous injection on day seven after tumor inoculation. 2 days after T-cell transfer, PLX4720 or vehicle alone was administered by oral gavage daily for 3 days. Luciferase imaging showing in vivo trafficking of OFL-expressing pmel-1 T cells on day five after T-cell transfer. Quantitative imaging analysis of transferred T cells at the tumor sites is summarized and expressed as the average of photon flux within ROI (a), Data shown are expressed as mean + SEM and are representative of two independent experiments with similar results. In addition, BRAF mutant A375 tumor-bearing mice were sacrificed 3 days after oral gavage of PLX4720, and tumors were resected and weighed. Tumors were homogenized and sonicated in lysis buffer containing protease inhibitors. Cleared tumor lysates after centrifugation were tested using protein array analysis (b).
Additionaly, Knight et al. utilized two relatively resistant syngeneic variants of BRAFV600E-driven mouse melanoma, SM1 and SM1WT1, and a transgenic mouse model of melanoma to illustrate the ability of the BRAF inhibitor, PLX4720, to reduce melanoma CCL2 production Interestingly, host CCR2 was demonstrated in the antitumor activity of PLX4720. While there was no obvious target molecules influenced with in the SM1WT1 tumor, there was an increase in the CD8/Treg ratio in the TILs with PLX4720 treatment. In addition, depleting CD8 + T cells, but not NK cells, were partially required for the therapeutic activity of PLX4720. Combination therapy of BRAF-directed therapies and anti-CCL2 or anti-CD137 antibodies demonstrated significant antitumor activity in these models supporting the therapeutic potential of combining BRAF inhibitors with immunotherapy .
Recently, a BRAF(V600E)/Pten-/ syngeneic tumor graft immunocompetent mouse model showed synergy of adding immune checkpoint blockade to BRAF inhibition . In this model, BRAF inhibition leads to a significant increase in intratumoral CD8+ T cell density and cytokine production, similar to effects of BRAF inhibition in patients. Furthermore, administration of anti-PD-1 or anti-PD-L1 blockade together with BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor infiltrating lymphocytes .
One manuscript has been published disputing possible synergy between BRAF-directed therapy and immunotherapy . This manuscript described work utilizing a murine model with conditional melanocyte-specific expression of BRAFV600E combined with Pten gene silencing which leads to development of melanoma with 100% penetrance, short latency, and lung and lymph node metastases. The mice are responsive to BRAF and MEK inhibition. In this paper, primary melanoma tumors were induced via topical Tamoxifen and were then treated with BRAF-directed therapy alone or in combination with immune checkpoint blockade. Of note, the induced melanomas showed histological and immune cell compartment similarities to human melanomas . However, unlike in humans [8, 34], there is a decrease in tumor resident lymphocytes in the setting of BRAF-directed therapy . Furthermore, the addition of CTLA4 blockade did not improve tumor growth control .
It is important to note that tumors generated in this model may be implanted into syngeneic C57BL/6 mice, suggesting a potential for a syngeneic subcutaneous tumor model . This is relevant as other groups (including our own) have used this approach with syngeneic subcutaneously implanted tumors and have demonstrated synergy with BRAF-directed therapy and immunotherapy . The syngeneic subcutaneously implanted tumor model in C57BL/6 may better recapitulate metastatic disease, though this is a hypothesis that clearly needs to be tested.
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