Reversal of pre-existing NGFR-driven tumor and immune therapy resistance

Abstract

Melanomas can switch to a dedifferentiated cell state upon exposure to cytotoxic T cells. However, it is unclear whether such tumor cells pre-exist in patients and whether they can be resensitized to immunotherapy. Here, we chronically expose (patient-derived) melanoma cell lines to differentiation antigen-specific cytotoxic T cells and observe strong enrichment of a pre-existing NGFR^hi population. These fractions are refractory also to T cells recognizing non-differentiation antigens, as well as to BRAF + MEK inhibitors. NGFR^hi cells induce the neurotrophic factor BDNF, which contributes to T cell resistance, as does NGFR. In melanoma patients, a tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance, and NGFR^hi tumor fractions are associated with immune exclusion. Lastly, pharmacologic NGFR inhibition restores tumor sensitivity to T cell attack in vitro and in melanoma xenografts. These findings demonstrate the existence of a stable and pre-existing NGFR^hi multitherapy-refractory melanoma subpopulation, which ought to be eliminated to revert intrinsic resistance to immunotherapeutic intervention.

Fig. 1. Melanoma fractions resistant to antigen-specific T cells.

a Graphic overview of the generation of T cell-resistant (TR) cell lines. b Colony formation assay of three parental cell lines (par) and their TR counterparts, treated with either control (Ctrl) or MART-1 T cells for 24 h in a 1:1 ratio and recovered for 3 days. Quantification in Supplementary Fig. 1a. c Tumor growth of melanoma cell lines D10 and the T cell-resistant counterpart, D10-TR. Randomization occurred on day 10 (indicated by dotted line). n = 7 per group except n = 6 for D10 + MART-1 T cells. Statistical analysis by Kruskal–Wallis test on day 21; *p < 0.05. d mRNA expression levels of MLANA in parental versus TR cell lines. Pooled data of six independent cell line pairs, lines indicate each paired parental and TR cell line. One experiment of three pooled technical replicates is shown; the data are reproduced in two independent replicates (available in Source data). e Cell viability after T cell attack of M009R.X1.CL cells for CDK^R24C and NY-ESO-1 TCRs. An experiment of two independent replicates with three technical replicates is shown (other replicate can be found in Source data). Statistical analysis by unpaired t-test; **p < 0.01. f Gene expression changes and average signature scores in TR cells vs. parental cells, normalized per cell line. Gene lists for different subtypes/cell states were derived from Tsoi et al. (see Methods section). g Flow cytometry quantification of AXL and NGFR expression in six matched cell lines. Error bars indicate S.D. of four independent replicates. Statistical analysis by Mann–Whitney test; *p < 0.05, ns not significant. h Immunohistochemistry analysis of NGFR on human melanoma, D10 tumors and D10-TR tumors. Scale bars indicate 50 μm. i FACSort lay-out of NGFR^low and NGFR^hi cells. j Quantification of cell viability after MART-1 T cell attack in three cell lines, similarly sorted as in i. Data of two independent replicates with two and three technical replicates, respectively. Statistical analysis by Mann–Whitney test; *p < 0.05, **p < 0.01. All error bars in this figure represent S.D. Source data are provided as a Source Data file.

Fig. 2. NGFR^hi melanomas are resistant to multiple therapies.

a Colony formation assay on IFNγ treatment on D10 and D10-TR cells. Tumor cells were treated for 7 days with IFNγ (which was refreshed on day 4) and stained with crystal violet. Quantification in b. b Quantification of IFNγ and TNF treatment for 7 days on D10 and D10-TR cells (medium was refreshed on day 4). Error bars represent S.D. of three independent replicates. Statistical analysis by unpaired t-test; *p < 0.05, ***p < 0.001. c Cytotoxicity assay in D10 parental versus TR cells for BRAF + MEKi. Titrations were performed with a 10:1 ratio of BRAFi:MEKi concentration. Error bars represent S.D. of three biological replicates; the experiment was performed in four independent replicates (as quantified in d). d Quantification of three cell lines for BRAF/MEKi sensitivity in parental versus TR lines. BRAF inhibitor dabrafenib was given at 10 nM and MEK inhibitor trametinib at 1 nM. Error bars represent S.D. of four independent replicates. Statistical analysis by Mann–Whitney test; *p < 0.05. e Cell count in parental and TR cells of the D10 cell line, treated continuously with 1000 nM of dabrafenib. Error bars represent S.D. of two independent replicates. Statistical analysis by Mann–Whitney test; *p < 0.05. f Average NGFR expression in PDX samples from melanoma. Error bars represent S.D. Statistical analysis by unpaired t-test. *p < 0.05. g NGFR immunohistochemistry score for patient samples prior to and on BRAF + MEK inhibition, divided in responders (R) and nonresponders (NR). Statistical analysis by Mann–Whitney, **p < 0.01. Source data are provided as a Source Data file.

Fig. 3. Pre-existing NGFR^hi cells display a stable phenotype.

a Relative MFI as assessed by flow cytometry for parental cell lines after a 24 h co-culture with MART-1 T cells in a 1:8 ratio. After 24 h, T cells were washed off and the medium was refreshed every 3 days. Data shown from three independent replicates. b Relative MFI as assessed by flow cytometry of TR cell lines compared with their parental counterparts over a period of 3 months without any T cell stimulation. c D10 and D10-TR clones were obtained by FACS-based single cell sorting and expansion, and after 2 months they were subjected to MART-1 T cells in a 1:1 ratio. The relative survival compared with parental D10 cells is shown, based on quantification of the corresponding colony formation assays (from two independent replicates). Statistical analysis by Mann–Whitney test by comparing all TR clones to all parental clones; *p < 0.05. d D10 tumors were treated at day 7 with Ctrl or MART-1 T cells in vivo, and at an average tumor volume of 400 mm³ individual mice were re-stratified to sequential BRAF + MEKi treatment (n = 6 per group; dabrafenib 30 mg/kg, trametinib 0.1 mg/kg). Error bars represent S.D. Statistical analysis by Mann–Whitney test on day 7; **p < 0.01. Source data are provided as a Source Data file.

Fig. 4. NGFR predicts immunotherapy resistance in melanoma patients.

a Heat map of the NGFR signature on a cohort of n = 95 melanoma PDX. b Gene Set Enrichment Analysis (GSEA) of NGFR signature (“upregulated in NGFR^hi”) on three parental vs. TR cell lines. c, d GSEA on two clinical datasets of patients treated with anti-PD-1 therapy. References for the source data of these databases can be found in the Methods section.

Fig. 5. NGFR^hi melanoma fractions show immune exclusion in patients.

a Examples of two melanomas from patients showing either high or low NGFR expression, and the corresponding CD3 and CD8 stainings for the same regions. Scale bars indicate 50 μm. Quantification in b, c. b, c CD3 and CD8 scores for NGFR^neg vs. NGFR^pos samples. Only tumor-infiltrating lymphocytes were scored and compared with the relative abundance of tumor cells. Error bars represent S.D. Statistical analysis by Mann–Whitney test; *p < 0.05. d Examples of two melanomas from patients that are heterogeneous for NGFR expression within each sample, and the corresponding CD3 stainings for the same regions (indicated by dotted lines). Scale bars indicate 100 μm. e MCP counter analysis on melanoma cohort of TCGA divided in top and bottom 25% NGFR-expressing tumors. Gene sets for “T cells” and “cytotoxic lymphocytes” are plotted. Error bars represent S.D. Statistical analysis by unpaired t-test. Source data are provided as a Source Data file.

Fig. 6. Genetic perturbation of NGFR modulates T cell sensitivity.

a Western blot analysis of indicated cell lines transduced with a control hairpin or one of two short hairpins targeting NGFR. Vinculin was used as a loading control. One blot of two independent replicates is shown (the other can be found in Source data). b Quantification of MART-1 T cell cytotoxicity in TR cell lines relative to Ctrl T cells (1:1 ratio tumor cell: T cell) in the presence of absence of shNGFR. Cells were loaded with 100nM MART-1 peptide prior to the assay, and cytotoxicity was normalized to shCtrl cell survival after MART-1 T cell attack. Error bars represent S.D. of four independent experiments with two technical replicates. Statistical analysis by Kruskal–Wallis test; *p < 0.05, ****p < 0.001. c Western blot analysis on D10 and M009R.X1.CL cells transduced with a control ORF or an NGFR-RFP ORF. GAPDH was used as a loading control. One blot of two independent replicates is shown (the other can be found in Source data). d Quantification of cell survival by flow cytometry after T cell attack in vitro in control or NGFR-ORF cells. Error bars represent S.D. of four independent experiments. Statistical analysis by Mann–Whitney, *p < 0.05. e mRNA expression of BDNF in parental and TR cells. Error bars represent S.D. of pooled analysis of three independent replicates. Analysis by unpaired t-test; *p < 0.05. f Quantification of cytotoxicity in TR cell lines relative to shCtrl cells after T cell attack (1:1 ratio tumor : T cell) in the presence or absence of shBDNF. Error bars represent S.D. of six independent experiments. Statistical analysis by Kruskal–Wallis test; *p < 0.05, **p < 0.01. Source data are provided as a Source Data file.

Fig. 7. Pharmacological reversal of NGFR^hi state restores T cell sensitivity.

a T cell titration in the presence or absence of tyrphostin (AG-879) in M009R.X1.CL-TR cells. b Quantification of MART-1 T cell cytotoxicity in TR cell lines relative to Ctrl T cells in the presence of absence of AG-879. Error bars represent S.D. of six independent experiments. Statistical analysis by Kruskal–Wallis test; **p < 0.01. c Western blot analysis on D10-TR cells treated with 250 nM of HSP90 inhibitor ganetespib for the indicated days. GAPDH was used as a loading control (see Source data for other replicate). d Quantification of NGFR expression by flow cytometry after 3 days of ganetespib treatment in vitro. Error bars represent S.D. of five independent experiments. Statistical analysis by Mann–Whitney, **p < 0.01. e Colony formation assay of D10-TR cells, treated with control (Ctrl) or MART-1 T cells in a 1:1 ratio and/or 250 nM ganetespib for 72 h. Quantification in f. f Quantification of cytotoxicity in TR cell lines relative to Ctrl T cells (1:1 ratio tumor cell: T cell) in the presence of absence of ganetespib. Two different sets of TCRs are shown. Error bars represent S.D. of six independent replicates. Statistical analysis by Mann–Whitney test; **p < 0.01. g Immunohistochemistry scoring of NGFR on D10-TR tumors after vehicle or 100 mg/kg ganetespib treatment in vivo during 6 days for n = 3 mice. Treatment was given on day 1 and day 4. Statistical analysis by unpaired t-test; *p < 0.05. h Tumor growth curve of D10-TR tumor cells after treatment with Ctrl T cells (n = 5), MART-1 T cells (n = 5), ganetespib 100 mg/kg + Ctrl T cells (n = 7), and ganetespib 100 mg/kg + MART-1 T cells (n = 8). Randomization, T cell injection, and start of ganetespib treatment occurred on day 7. Ganetespib was given twice weekly. Error bars represent S.D. Statistical analysis by Kruskal–Wallis test; ns not significant, *p < 0.05, **p < 0.01. i Survival curve belonging to h. Statistical analysis by Log-Rank Mantel–Cox test, **p < 0.01. Source data are provided as a Source Data file.