Intermittent BRAF inhibition in advanced BRAF mutated melanoma results of a phase II randomized trial

Combination treatment with BRAF (BRAFi) plus MEK inhibitors (MEKi) has demonstrated survival benefit in patients with advanced melanoma harboring activating BRAF mutations. Previous preclinical studies suggested that an intermittent dosing of these drugs could delay the emergence of resistance. Contrary to expectations, the first published phase 2 randomized study comparing continuous versus intermittent schedule of dabrafenib (BRAFi) plus trametinib (MEKi) demonstrated a detrimental effect of the “on−off” schedule. Here we report confirmatory data from the Phase II randomized open-label clinical trial comparing the antitumoral activity of the standard schedule versus an intermittent combination of vemurafenib (BRAFi) plus cobimetinib (MEKi) in advanced BRAF mutant melanoma patients (NCT02583516). The trial did not meet its primary endpoint of progression free survival (PFS) improvement. Our results show that the antitumor activity of the experimental intermittent schedule of vemurafenib plus cobimetinib is not superior to the standard continuous schedule. Detection of BRAF mutation in cell free tumor DNA has prognostic value for survival and its dynamics has an excellent correlation with clinical response, but not with progression. NGS analysis demonstrated de novo mutations in resistant cases.

Targeted BRAF inhibitors: Immunological effects and combination with immunotherapy

<p>Metastatic melanoma is the most aggressive form of skin cancer, associated with a poor prognosis, and the incidence worldwide is increasing. Recently, selective mutant BRAF inhibitors and checkpoint blockade immunotherapy have advanced clinical treatment of metastatic melanoma. However, efficacy of these therapies individually is limited. Combining treatments may allow BRAF inhibition to augment immunotherapy by increasing tumour antigen availability and improving immune system targeting of tumours. The success of this approach depends upon fully elucidating immunological interactions of BRAF inhibitors, and optimizing combination strategies. To study the immunological effects of BRAF inhibitors and their combination with immunotherapy, novel murine BrafV600E Pten-/- Cdkn2a cell lines were characterized. These were found to be moderately sensitive to BRAF inhibition compared with the widely used human BRAFV600E cell lines A375 and SK-mel-5. In vitro targeted BRAF inhibition was shown to induce cell death through apoptosis, and partially reverse melanoma-mediated immunosuppression by human melanoma cell lines. Utilising subcutaneously injected syngeneic, murine BRAFV600E cell lines, the BRAF inhibitor PLX4720 was shown to decrease tumour growth in vivo. Host immune involvement in BRAF inhibitor efficacy was determined by comparing PLX4720 treatment in NOD/Scid and C57BL/6 mice. PLX4720 control of tumour growth was significantly less effective in immunocompromised mice, resulting in reduced survival advantage. These findings demonstrate that the anti-tumour effects of mutant BRAF inhibitors are partially immune dependent, although the nature of this immune involvement remains to be defined. It was further shown that BRAFV600E inhibition directly affected immune responses. In vitro, both human and murine T cell activation were boosted by low concentrations of mutant BRAF inhibitors. This was confirmed in vivo, with antigen-specific T cell proliferation significantly increased by PLX4720 treatment. The final chapters of this thesis explored the combination of active immunotherapy with targeted BRAF inhibition. A vaccine was devised that consisted of irradiated, autologous tumour cells loaded with the adjuvant α-galactosylceramide. This vaccine was shown to be effective in both prophylactic and therapeutic settings in a BRAFV600E melanoma model. Mechanistically, vaccine increased effector T cell responses and decreased frequencies of Tregs. Vaccine efficacy was CD4⁺ T cell-dependent, and did not require CD8⁺ T cells. Combination of vaccine with targeted BRAF inhibition was investigated in different settings. A combination therapy strategy was developed that achieved additive, but not synergistic benefit. Additionally, targeting specific aspects of the tumour microenvironment that may confer tumour resistance to BRAF inhibitor-mediated cell death was investigated. Both depletion of Tregs and inhibition of TNFα were explored, but did not result in a significant improvement in therapy. In summary, the studies undertaken in this thesis demonstrate that BRAF inhibitors can augment vaccine-induced T cell responses. Moreover, this research revealed the anti-tumour efficacy of BRAFV600E inhibition is partially immune dependent and can be improved by combination with active immunotherapy. These discoveries generated a combination therapy strategy with improved efficacy over single agent treatment. Further studies are needed to realise the full potential of this combination therapy approach, and achieve a synergistic benefit.</p>

Targeted BRAF inhibitors: Immunological effects and combination with immunotherapy

<p>Metastatic melanoma is the most aggressive form of skin cancer, associated with a poor prognosis, and the incidence worldwide is increasing. Recently, selective mutant BRAF inhibitors and checkpoint blockade immunotherapy have advanced clinical treatment of metastatic melanoma. However, efficacy of these therapies individually is limited. Combining treatments may allow BRAF inhibition to augment immunotherapy by increasing tumour antigen availability and improving immune system targeting of tumours. The success of this approach depends upon fully elucidating immunological interactions of BRAF inhibitors, and optimizing combination strategies. To study the immunological effects of BRAF inhibitors and their combination with immunotherapy, novel murine BrafV600E Pten-/- Cdkn2a cell lines were characterized. These were found to be moderately sensitive to BRAF inhibition compared with the widely used human BRAFV600E cell lines A375 and SK-mel-5. In vitro targeted BRAF inhibition was shown to induce cell death through apoptosis, and partially reverse melanoma-mediated immunosuppression by human melanoma cell lines. Utilising subcutaneously injected syngeneic, murine BRAFV600E cell lines, the BRAF inhibitor PLX4720 was shown to decrease tumour growth in vivo. Host immune involvement in BRAF inhibitor efficacy was determined by comparing PLX4720 treatment in NOD/Scid and C57BL/6 mice. PLX4720 control of tumour growth was significantly less effective in immunocompromised mice, resulting in reduced survival advantage. These findings demonstrate that the anti-tumour effects of mutant BRAF inhibitors are partially immune dependent, although the nature of this immune involvement remains to be defined. It was further shown that BRAFV600E inhibition directly affected immune responses. In vitro, both human and murine T cell activation were boosted by low concentrations of mutant BRAF inhibitors. This was confirmed in vivo, with antigen-specific T cell proliferation significantly increased by PLX4720 treatment. The final chapters of this thesis explored the combination of active immunotherapy with targeted BRAF inhibition. A vaccine was devised that consisted of irradiated, autologous tumour cells loaded with the adjuvant α-galactosylceramide. This vaccine was shown to be effective in both prophylactic and therapeutic settings in a BRAFV600E melanoma model. Mechanistically, vaccine increased effector T cell responses and decreased frequencies of Tregs. Vaccine efficacy was CD4⁺ T cell-dependent, and did not require CD8⁺ T cells. Combination of vaccine with targeted BRAF inhibition was investigated in different settings. A combination therapy strategy was developed that achieved additive, but not synergistic benefit. Additionally, targeting specific aspects of the tumour microenvironment that may confer tumour resistance to BRAF inhibitor-mediated cell death was investigated. Both depletion of Tregs and inhibition of TNFα were explored, but did not result in a significant improvement in therapy. In summary, the studies undertaken in this thesis demonstrate that BRAF inhibitors can augment vaccine-induced T cell responses. Moreover, this research revealed the anti-tumour efficacy of BRAFV600E inhibition is partially immune dependent and can be improved by combination with active immunotherapy. These discoveries generated a combination therapy strategy with improved efficacy over single agent treatment. Further studies are needed to realise the full potential of this combination therapy approach, and achieve a synergistic benefit.</p>

Detection of clinical progression through plasma ctDNA in metastatic melanoma patients: a comparison to radiological progression

Background The validity of circulating tumour DNA (ctDNA) as an indicator of disease progression compared to medical imaging in patients with metastatic melanoma requires detailed evaluation. Methods Here, we carried out a retrospective ctDNA analysis of 108 plasma samples collected at the time of disease progression. We also analysed a validation cohort of 66 metastatic melanoma patients monitored prospectively after response to systemic therapy. Results ctDNA was detected in 62% of patients at the time of disease progression. For 67 patients that responded to treatment, the mean ctDNA level at progressive disease was significantly higher than at the time of response (P < 0.0001). However, only 30 of these 67 (45%) patients had a statistically significant increase in ctDNA by Poisson test. A validation cohort of 66 metastatic melanoma patients monitored prospectively indicated a 56% detection rate of ctDNA at progression, with only two cases showing increased ctDNA prior to radiological progression. Finally, a correlation between ctDNA levels and metabolic tumour burden was only observed in treatment naïve patients but not at the time of progression in a subgroup of patients failing BRAF inhibition (N = 15). Conclusions These results highlight the low efficacy of ctDNA to detect disease progression in melanoma when compared mainly to standard positron emission tomography imaging.

Rapid signaling reactivation after targeted BRAF inhibition predicts the proliferation of individual melanoma cells from an isogenic population

Cancer cells within tumors display a high degree of phenotypic variability. This variability is thought to allow some of the cells to survive and persist after seemingly effective drug treatments. Studies on vemurafenib, a signaling inhibitor that targets an oncogenic BRAF mutation common in melanoma, suggested that cell-to-cell variation in drug resistance, measured by long-term proliferation, originates from epigenetic differences in gene expression that pre-exist treatment. However, it is still unknown whether reactivation of signaling downstream to the inhibited BRAF, thought to be a key step for resistance, is heterogeneous across cells. While previous studies established that signaling reactivation takes place many hours to days after treatment, they monitored reactivation with bulk-population assays unsuitable for detecting cell-to-cell heterogeneity. We hypothesized that signaling reactivation is heterogeneous and is almost instantaneous for a small subpopulation of resistant cells. We tested this hypothesis by monitoring signaling dynamics at a single-cell resolution and observed that despite highly uniform initial inhibition, roughly 15% of cells reactivated signaling within an hour of treatment. Moreover, by tracking cell lineages over multiple days, we established that these cells indeed proliferated more than neighboring cells, thus establishing that rapid signaling reactivation predicts long-term vemurafenib resistance.

PERK mediates resistance to BRAF inhibition in melanoma with impaired PTEN

Targeting mutant BRAF in patients with melanomas harboring this oncogene has been highly successful as a first-line treatment, but other mutations may affect its efficacy and alter the route of acquired resistance resulting in recurrence and poor prognosis. As an evolving strategy, melanoma treatment needs to be expanded to include targets based on newly discovered emerging molecules and pathways. We here show that PERK plays a critical role in BRAF inhibitor-acquired resistance in melanoma with impaired PTEN. Inhibition of PERK by either shRNA or a pharmacological inhibitor blocked the growth of BRAF inhibitor-resistant melanoma with impaired PTEN in vitro and in vivo, suggesting an effective approach against melanomas with mutant BRAF and PTEN deficiency. Our current findings, along with our previous discovery that the AXL/AKT axis mediates resistance to BRAF inhibition in melanoma with wild-type PTEN, provide new insights toward a strategy for combating BRAF inhibition-acquired resistance in BRAF mutant melanoma with different PTEN statuses.