Decoding molecular programs in melanoma brain metastases

Abstract

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient’s prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.

MET receptor activation by stromal cells serves as promising target in melanoma brain metastases

“…Co-staining revealed accumulation of CD3 + T cells as well as of Iba1 high TAMs (Figure 2b). Ranking of MBM regarding levels of ITGB7 expression showed co-occurrence in the expression of CD4, CD274, Sushi Domain Containing 3 (SUSD3) and ITGB7 level (Figure 2c) and validated a possible, previously observed 23 correlation of ITGB7 and SUSD3. Moreover ITGB7, SUSD3 and APBB1IP showed expression across different immune cell types except for monocytes and NK cells (Supplementary figures 2c-f).…”

“…We therefore suggest that the activation of MET- or STAT3-mediated signaling processes or those related to stress/senescence or inflammation strongly depend on the composition of the tumor microenvironment, likely determining the response to therapeutic interventions. Although infiltration of TAMs is not evident in all MBM, microglia infiltration seems to be an early occurring process observed ∼21d after intracranial injection of BMCs into brains of immune compromised Crl:CD1-Foxn1 nu mice 23 (Figure 3b). Moreover, we observed activation of Stat3 signaling in tumor adjacent cells (Figure 3b), suggesting that brain microenvironmental cells are activated after a short time of tumor-stroma interaction and establish an inflammatory environment.…”

“…Since no data on TAM-infiltrated MBM are available, we performed comparative methylome and transcriptome profiling of Iba1 high (n=5-10) and Iba1 low (n=2-6) tumors and identified a set of 417 differentially methylated genomic regions (DMRs) that corresponded to 294 MBM expressed genes (Figure 1g) a core set of markers (n=31) sufficient to split tumors (Figure 1h; Supplementary tables 2, 3). Among them, we identified the integrin family member and gut-homing receptor ITGB7 -which we described in our previous study as a distinguishing mark between BRAF and NRAS mutant MBM 23 - and APBB1IP (amyloid b precursor protein-binding family b member 1 interacting protein). Both are associated with better prognosis in patients with colorectal cancer 27,28 and clustered with known TAM-associated genes such as P2RY12 and AIF1 (Figure 1h).…”

“…Briefly, all brain metastases-derived cell lines (BMCs) and conventional melanoma cell lines were kept at 37°C/ 5% CO 2 and 95% humidity in cell culture medium (DMEM, 4.5 g/L glucose, stabilized glutamine/GlutaMax, pyruvate, Gibco/ThermoFisher) supplemented with 10% fetal bovine (FBS, Gibco) serum and 1% penicillin/streptomycin (P/S) (Gibco/ThermoFisher) and routinely passaged. BMCs were established from intraoperative tumors as previously reported 23 .…”

“…Isolation of total RNA from snap frozen tumors and RNA sequencing was performed as previously reported 23 . Briefly, 100 ng of total RNA was used for library preparation with TruSeq Stranded total RNA Sample Preparation-Kit and Ribo-Zero Gold Kit (Illumina).…”