BRAF inhibitors and their immunological effects in malignant melanoma

Figures & data

Figure 1. The mitogen-activated signaling pathway, from left to right: a) normal function of MAPK pathway signaling; b) BRAF^V600E mutation leading to constituently activated MAPK signaling and enhanced pathogenesis of melanoma; c) targeting of MAPK pathway, with BRAFi and MEKi, reducing melanoma pathogenicity; and d) mechanisms by which resistance can develop. Created with BioRender.com.

Figure 2. Potential immunological effects of BRAFi, BRAFi resistance, and mechanisms to therapeutically enhance BRAFi: a) The immunosuppressive TME created in BRAF mutant melanoma includes the recruitment and expansion of immunosuppressive cells, effector cells with reduced cancer killing potency and the secretion of factors by immune cells that can increase cancer growth and angiogenesis. b) The proinflammatory TME created by BRAFi includes increased effector immune cell recruitment and expansion, reduced immunosuppressive immune cell recruitment, and increased ability of immune effector cells to trigger cancer killing. c) As BRAFi resistance develops, the reversal of BRAFi immune effects is observed. d) There are multiple mechanisms by which BRAFi resistance can be overcome using current and future therapies. These include depleting immunosuppressive cells such as tumor-associated macrophages (TAMs), targeting angiogenesis and growth promoting vascular endothelial growth factor (VEGF), targeting immune exhaustion molecules with checkpoint inhibitors (PD-1/PD-L1), and enhancing cancer killing with adoptive cell therapy (ACT), including the use of chimeric antigen receptor T cell therapies (CAR-T), as well as stimulating effector cells, for example, by using toll-like receptor agonists (TLR). Created with BioRender.com.

Table 1. BRAFi effect on immune function: preclinical data of the effect of BRAFi on the tumor microenvironment, specifically changes in immune mechanisms and pathways. TILS – tumor infiltrating lymphocytes; NK cells – natural killer cells; T-regs – regulatory T cells; MDSCs – myeloid-derived suppressor cells; APCs- antigen-presenting cells

BRAFi effect of immune function	Findings	Model type	Papers/Refs
Immune stimulatory factors	Increased mRNA expression of IL-12a, IL-12b, IL-15, IL-18	In vivo mouse model	Bellmann et al. [50]
	Increased mRNA expression of chemokines CCL2, CCL3, CCL4, CXCL9, CXCL10	In vivo mouse model	Bellmann et al. [50]
	Increased production of IFNγ	In vivo mouse model	Ho et al. [43]
Immunosuppressive factors	Decreased IL-6, IL-8	Tissue samples from patients	Frederick et al. [46]
	Decreased PD-L1, IL-1, IL-8, VEGFA	In vitro human cell lines and in vivo mouse model	Liu et al. [60]
	Decreased IL-7, CX3CL1, GCSF, CXCL1, TGFa2, IL-8, VEGF, IFNα	Human cell lines	Whipple et al. [55]
	Decreased VEGF	Tissue samples from patients	Liu et al. [59]
Immune cell infiltration	Increased TILs	Tissue samples from patients	Frederick et al. [46]
		In vivo mouse model	Knight et al. [41]
		Tissue samples from patients	Wilmott et al. [49]
		In vivo mouse model + one patient follow-up throughout treatment	Cooper et al. [74]
		In vivo mouse model	Ho et al. [43]
	Increased tumor infiltrating NK cells	In vivo mouse model	Bellmann et al. [50]
		In vivo mouse model	Knight et al [41]
	Increased CD8:CD4 ratio in TILs	In vivo mouse model + one patient follow-up throughout treatment	Cooper [74]
	Decreased T-regs	In vivo mouse model	Knight et al. [41]
	Decreased MDSCs	Blood samples from patients	Schilling et al. [53,54]
		In vivo mouse model	Ho et al. [43]
Immune cell function	Increased melanoma antigen expression MART1, TYRP1, TYRP2, GP100 Increased MHC class I and II	Tissue samples from patients	Frederick et al. [46]
		In vivo mouse model	Bellmann et al. [50]
		In vitro human cell lines and in vivo mouse model	Liu et al. [60]
		Human cell lines	Whipple et al. [55]
		Human cell lines	Bradley et al. [64]
	Increased markers of T cell cytotoxicity: increased perforin, granzyme B	Tissue samples from patients	Frederick et al. [46]
		Tissue samples from patients	Wilmott et al. [49]
	Increased maturation of APCs	In vivo mouse model	Ho et al. [43]
	Increased expression of CD40L on CD4	In vivo mouse model	Ho et al. [43]
Immune cell exhaustion	Increased PD-1, PD-L1 and TIM3	Tissue samples from patients	Frederick et al. [46]
	Increased PD-L1 and PD-L2	In vivo mouse model	Cooper et al. [74]

Table 2. Clinical trials of combination therapies in metastatic melanoma with BRAFi/MEKi with another therapeutic intervention

	Study (NCT)	Phase	Clinical Setting			Line of Treatment	Arms	Primary and Secondary End Points	Outcome
BRAFi/MEKi and ICI – Upfront Combination	Ribas et al. (73)	1	BRAF^V600E mutant positive metastatic melanoma			First line	Arm 1 (n = 6): Vemurafenib (high dose) run in + Ipilimumab Arm 2 (n = 6): Vemurafenib (low dose) + Ipilimumab	Evaluate safety and dose administration	Discontinued due to hepatotoxicity
	Minor et al. (74) NCT01767454	1/2	BRAF^V600E mutant positiveunresectable or metastatic melanoma			Unspecified	Arm 1: Dabrafenib + Ipilimumab Arm 2: Dabrafenib + Trametinib + Ipilimumab	Evaluate safety of combinations	Discontinued due to intestinal perforation (n = 2) in the triplet arm
	KEYNOTE-022 NCT02130466	1/2	Metastatic melanoma			Unspecified	BRAF mutant: Dabrafenib + Trametinib + Pembrolizumab BRAF wild type (BRAF^WT) continuous administration: Trametinib 1 month run in + Dabrafenib + Pembrolizumab BRAF^WT intermittent administration: Trametinib intermittent + Pembrolizumab	Number of participants with doselimiting toxicities Objective response rate in those without the BRAF^V600E/K mutations PFS in those with the mutation	Phase 1: ORR 67% in BRAF-mutant Phase 2: (Dabrafenib + Trametinib + Pembrolizumab versus Dabrafenib + Trametinib + placebo) in BRAF mutant: ORR 63% versus 72% 73% of patients with grade 3–4 treatment related toxicities
	TRIDeNT NCT02910700	2	BRAF^V600E mutant positive unresectable or metastatic melanoma (stage III/stage IV)			Unspecified	Binimetinib: MEK 1/2 inhibitor Arm 1: Dabrafenib + Trametinib + Nivolumab Arm 2: Trametinib + Nivolumab Arm 3: Binimetinib + Encorafenib + Nivolumab	Objective Response Rate Secondary: Incidence of adverse events Complete response Partial response	ORR 91% 21% discontinued study due to toxicity (hepatitis + nephritis)
	COMBI-I, NCT02967692	3, part 1	Previously untreated patients with unresectable locally advanced disease or metastatic melanoma BRAF^V600E mutant positive			First line	Spartiluzimab: anti-PD-1 antibody Arm 1: Spartiluzimab + Dabrafenib + Trametinib Arm 2: Pembroluzimab + Dabrafenib + Trametinib	Safety run in Part 1 – evidence of dose limiting toxicities Biomarker cohort Randomized PFS	ORR 100% 22% discontinued Spartilizumab due to toxicity (hepatitis + transaminitis)
	TRILOGY, NCT02908672 (IMspire150)	3 (double blind, randomized)	Previously untreated patients with unresectable locally advanced disease or metastatic melanoma BRAF^V600E mutant positive			First line	Placebo Arm: Vemurafenib + Cobimetinib (n = 256) Experimental Arm: Vemurafenib + Cobimetinib + Atezolizumab (n = 258)	PFS Secondary: Percentage of participants with objective response rate Duration of response Overall Survival	Active HR = 0.72 (p = 0.025)
BRAFi/MEKi and ICI – Lead In	NCT01656642	1b (open label)	BRAF^V600E mutant positive melanoma			First line	Arm 1: Vemurafenib + Cobimetinib 1 month run in followed by Atezolizumab Arm 2: Vemurafenib + Cobimetinib + Atezolizumab	Percentage of Participants with Dose Limiting Toxicities Percentage of participants with adverse events Secondary: Pharmacokinetics Measurements Percentage of Participants with Objective Response	Enrollment complete ORR 85.3% 44.1% of patients with grade 3–4 treatment related adverse events
	NCT02027961	1	BRAF^V600E mutant positive			Unspecified	Durvalumab: anti-PD-L1 antibody BRAF mutant: Dabrafenib + Trametinib + Durvalumab BRAF^WT concurrent: Trametinib + Durvalumab BRAF^WT sequential: Trametinib →Durvalumab	Number of participants with dose-limiting toxicities and treatment- related adverse events Secondary: Percentage of Participants with Objective Response Duration of Response PFS	ORR 76% in BRAF mutant ORR 21% in BRAF^WT concurrent ORR 50% BRAF^WT sequential 39% of patients with grade 3–4 treatment related toxicities in BRAF – mutant, 40% in BRAF^WTconcurrent, 17% in BRAF^WTsequential
BRAFi/MEKi and ICI – Cycling/Sequencing	IMPemBra (NCT02625337)	2	Stage IV BRAF^V600E or BRAF^V600K positive metastatic melanoma			First Line (n = 32)	Arm 1: Pembrolizumab Arm 2: Pembrolizumab + Dabrafenib + Trametinib (short) Arm 3: Pembrolizumab + Dabrafenib + Trametinib (intermediate) Arm 4: Pembrolizumab + Dabrafenib + Trametinib (long)	Safety of different schemes of continuous/intermittent Dabrefinib + Trametinib during treatment with Pembrolizumab Feasibility of different schemes of continuous/intermittent Dabrefinib-Trametinib Secondary: Determine PFS Determine rates of response Determine long term toxicities	After a median follow-up of 17.4 months, the median PFS of patients treated with PEM monotherapy was 10.6 months compared to 27.0 months for patients treated with PEM and short-term/intermittent Dabrafenib + trametinib (p = 0.13)
	DREAMseq (NCT02224781)	3	Locally advanced unresectable melanoma or stage IV melanoma			Unspecified	Arm A: Nivolumab + Ipilimumab (induction), Nivolumab (maintenance) Arm B: Dabrafenib + Trametinib, followed by Nivolumab + Ipilimumab Arm C: Dabrafenib + Trametinib Arm D: Nivolumab + Ipilimumab (induction), Nivolumab (maintenance)	Overall survival rate Secondary: PFS Objective Response Rate Toxicity Rate for irAEs	Active, recruiting
	SECOMBIT (NCT 02631447)	2	Metastatic melanoma withtheBRAF^V600E mutation			Unspecified	BRAFi: LGX818 MEKi: MEK162 PD = progressive disease Arm A: LGX818 + MEK162 until PD, followed by Nivolumab + Ipilimumab Arm B: Nivolumab + Ipilimumab until PD, followed by LGX818 + MEK162 Arm C: LGX818 + MEK162 for 8 weeks, Nivolumab + Ipilimumab until PD, then LGX818 + MEK162	Overall Survival Secondary: Total PFS, best overall response rate and duration of response	Active, not recruiting
	NEOTRIO (NCT02858921)	2	BRAF^V600 mutantresectable Stage III B/ III C melanoma			Unspecified	Three Arms: Arm 1: Dabrafenib + Trametinib, then Pembrolizumab Arm 2: Dabrafenib + Trametinib + Pembrolizumab (6 w), then Pembrolizumab Arm 3: Pembrolizumab	Pathological Response Rate Secondary: Objective Clinical Response Rate Relapse Free Survival Overall Survival	Active, not recruiting
BRAFi/MEKi andantiangiogenic agent	NCT01495988	2	Stage IV BRAF^V600E positive melanoma		First line		Arm 1: Vemurafenib + Cobimetinib + Placebo Arm 2: Vemurafenib + Cobimetinib + Bevacizumab	Maximum tolerated dose Median PFS Secondary: Overall Survival + Response Rates	Terminated – slow accrual/toxicity
	NCT03175432	2	Untreated melanoma withbrain metastasis		First Line		Arm 1: Bevacizumab + Atezoluzimab + Placebo Arm 2: Bevacizumab + Atezolizumab + Cobimetinib	Objective intracranial response rate Safety and tolerability of the arms in trial Secondary: Incidence of adverse events Overall response rates Duration of response	Active, recruiting
BRAFi/MEKi and cell-based therapy	NCT03455764	1 + 2	AdvancedmelanomaBRAF^V600E/K positive	Second line (following BRAFi/MEKi treatment)			Lacnotuzumab (MCS110): Colony-stimulating factor-1 (CSF-1) inhibitor antibody Lacnotuzumab + Dabrafenib + Trametinib	Dose-Limiting Toxicity Secondary: Overall Response Rate Complete Response Rate	Active, not recruiting
	NCT03101254	1 + 2	Advanced melanoma BRAF^V600E/K positive	First line			LY3022855: Colony-stimulating factor-1 receptor (CSF-1 R) inhibitor LY3022855 + Vemurafenib and Cobimetinib	PFS Secondary: Side Effects from Therapy Overall Response Rate	Active, not recruiting

NCT = clinicaltrials.gov number.

Table

BRAFi					BRAF inhibitors
MEKi					MEK inhibitors
ICI					immune checkpoint inhibitors
MAPK					mitogen-activated signalling pathway
MAPKi					mitogen-activated signalling pathway inhibitors
PFS			Progression free survival
TME			tumour microenvironment
OS			overall survival
MDSC			myeloid derived suppressor cells
irAEs			immune related adverse events
trAEs			treatment-related adverse events
T-reg	regulatory T cells
TAMs	tumour associated macrophages
VEGF	vascular endothelial growth factor
APCs	antigen presenting cells
CTLs		cytotoxic T cells
NK cells		natural killer cells
DCs				dendritic cells
MHC-I				major histocompatibility complex
TILs				tumour infiltrating lymphocytes
ACT				adoptive cell therapy
CAR-T				chimeric antigen T-cell therapy

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