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>

Melanomagenesis driven by a kinase-dead mutant BRAF lacking codons 600-618

While considerable success has been achieved with treating BRAF-V600E mutant tumors in several organ systems, tumors expressing non-V600E BRAF mutations remain a significant clinical challenge. Such atypical BRAF mutations are classified based on their kinase activity, RAS-dependence and their requirement for dimerization to function. We identified an unusually large in-frame deletion in a melanoma patient, encompassing codons 600-618 in the kinase domain. This patient experienced rapid disease progression when treated with dual BRAF/MEK inhibition. To determine whether and how such a large in-frame BRAF deletion might be activating, we ectopically expressed it in HEK293 cells and monitored BRAF pathway activation and response to BRAF and MEK inhibitors. We demonstrate that this mutation results in a kinase-deficient BRAF mutant which, nevertheless, activates MEK/ERK signaling in a dimerization-dependent manner, placing this mutation among the class III BRAF mutants.

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.

Ultrasensitive Detection of BRAF Mutations in Circulating Tumor DNA of Patients With Metastatic Thyroid Cancer

Background: Liquid biopsy is a promising technology that can offer various advantages for molecular testing over tissue-based approaches. Most studies trying to address the utility of liquid biopsy in thyroid cancer have failed so far to achieve satisfactory rates of detection of relevant mutations. In this study, we examined a newly developed approach for ultrasensitive detection of oncogenic mutations in thyroid cancer using BRAF mutation as a proof-of-concept. In an exploratory analysis, we also correlated our findings with clinical outcomes and with levels of standard of care biomarkers. Methods: We included a group of patients with metastatic thyroid carcinoma. Cell free DNA (cfDNA) was isolated from an average of 2 ml of plasma and from matched formaldehyde fixed paraffin tissue blocks (FFPB) that were obtained from prior surgery. Extracted DNA was subject to preamplification of mutant copies using Q5 High-Fidelity PCR kit. Digital droplet PCR was performed on pre-amplified purified DNA where BRAF mutated allele frequencies (AF) were measured using BioRad ddPCR Qx200. Results: Thirty-three patients were included in our study with a median age at diagnosis of 62. Our method achieved a sensitivity of detection of 47.6% and a specificity of 80%. Mutant BRAF V600E was detected in cfDNA of 54.5% of patients (n=18) compared to 80.8% in isolated DNA from matched FFPB. Median overall survival (OS) was shorter in patients with wild type (WT) BRAF in both ctDNA and tissue (127m vs 218m, p=0.015; 116m vs 223m, p=0.004). Thyroglobulin (Tg) levels did not correlate with BRAF mutations either quantitatively or qualitatively. In the papillary thyroid carcinoma-classic variant cohort (n=20), however, patients with cfDNA mutant BRAF were more likely to have elevated Tg (90.9% versus 44.4% respectively, p=0.05). Conclusions: Our study provided a proof of concept for a newly developed technique to provide high sensitivity of mutation detection in thyroid cancer. The achieved sensitivity of detection is the highest to date using liquid biopsy in this tumor type. While we addressed only BRAF mutations in our study, the same approach can potentially be used for other mutations as well, likely changing the paradigm for use of liquid biopsy in thyroid cancer.

Microsatellite instability (MSI-H) is associated with a high immunoscore but not with PD-L1 expression or increased survival in patients (pts.) with metastatic colorectal cancer (mCRC) treated with oxaliplatin (ox) and fluoropyrimidine (FP) with and without bevacizumab (bev): a pooled analysis of the AIO KRK 0207 and RO91 trials

Introduction In a retrospective analysis of two randomized phase III trials in mCRC patients treated first line with oxaliplatin, fluoropyrimidine with and without Bevacizumab (the AIO KRK 0207 and R091 trials) we evaluated the association of high microsatellite instability (MSI-H), immunoscore (IS) and PD-L1 expression in relation to overall survival (OS). Methods In total, 550 samples were analysed. Immunohistochemical analysis of the MMR proteins and additionally fragment length analysis was performed, molecular examinations via allele-discriminating PCR in combination with DNA sequencing. Furthermore PD-L1 and IS were assessed. Results MSI-H tumors were more frequent in right sided tumors (13.66% vs. 4.14%) and were correlated with mutant BRAF (p = 0.0032), but not with KRAS nor NRAS mutations (MT). 3.1% samples were found to be PD-L1 positive, there was no correlation of PDL1 expression with MSI-H status, but in a subgroup analysis of MSI-H tumors the percentage of PD-L1 positive tumors was higher than in MSS tumors (9.75% vs. 2.55%). 8.5% of samples showed a positive IS, MSI-H was associated with a high IS. The mean IS of the pooled population was 0.57 (SD 0.97), while the IS of MSI-H tumors was significantly higher (mean of 2.4; SD 1.4; p =< 0.0001). Discussion Regarding OS in correlation with MSI-H, PD-L1 and IS status we did not find a significant difference. However, PD-L1 positive mCRC tended to exhibit a longer OS compared to PD-L1 negative cancers (28.9 vs. 22.1 months).

Mutant-selective degradation by BRAF-targeting PROTACs

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable with degradation-based approaches by targeting mutant BRAF-driven cancers while sparing BRAFWT, providing an anti-tumor drug modality that expands the therapeutic window.

No Impact of NRAS Mutation on Features of Primary and Metastatic Melanoma or on Outcomes of Checkpoint Inhibitor Immunotherapy: An Italian Melanoma Intergroup (IMI) Study

Aims: It is debated whether the NRAS-mutant melanoma is more aggressive than NRAS wildtype. It is equally controversial whether NRAS-mutant metastatic melanoma (MM) is more responsive to checkpoint inhibitor immunotherapy (CII). 331 patients treated with CII as first-line were retrospectively recruited: 162 NRAS-mutant/BRAF wild-type (mut/wt) and 169 wt/wt. We compared the two cohorts regarding the characteristics of primary and metastatic disease, disease-free interval (DFI) and outcome to CII. No substantial differences were observed between the two groups at melanoma onset, except for a more frequent ulceration in the wt/wt group (p = 0.03). Also, the DFI was very similar in the two cohorts. In advanced disease, we only found lung and brain progression more frequent in the wt/wt group. Regarding the outcomes to CII, no significant differences were reported in overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) or overall survival (OS) (42% versus 37%, 60% versus 59%, 12 (95% CI, 7–18) versus 9 months (95% CI, 6–16) and 32 (95% CI, 23–49) versus 27 months (95% CI, 16–35), respectively). Irrespectively of mutational status, a longer OS was significantly associated with normal LDH, <3 metastatic sites, lower white blood cell and platelet count, lower neutrophil-to-lymphocyte (N/L) ratio. Our data do not show increased aggressiveness and higher responsiveness to CII in NRAS-mutant MM.