Immunotherapy Resistance by Inflammation-Induced Dedifferentiation

Abstract

A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced in vitro by treatment with MART1 T cell receptor-expressing T cells or with TNFα, and that the phenotype is reversible with withdrawal of inflammatory stimuli. This supports the hypothesis that acquired resistance to cancer immunotherapy can be mediated by inflammation-induced cancer dedifferentiation.Significance: We report a patient whose metastatic melanoma underwent inflammation-induced dedifferentiation as a resistance mechanism to ACT to the MART1 antigen. Our results suggest that future melanoma ACT protocols may benefit from the simultaneous targeting of multiple tumor antigens, modulating the inflammatory response, and inhibition of inflammatory dedifferentiation-inducing signals. Cancer Discov; 8(8); 935-43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.

Trial registration: ClinicalTrials.gov NCT00910650.

Figure 1

Progressive tumors after ACT demonstrate loss of melanocytic antigens. (A) Overall treatment course for this patient. The patient was treatment naïve at the time of initiation of the ACT protocol. The patient underwent leukophoresis, and PBMCs were used to manufacture T cells for ACT and DCs for use in a MART-1_26–35 pulsed DC vaccine. The patient underwent non-myeloablative conditioning with cyclophosphamide and fludarabine started at day −7 prior to ACT. He received the DC vaccine on days +1, +14 and +30 after ACT. Several follow-up PET/CT scans were performed, initially showing disease regression at day +35 and eventual progression by day +84. (B)–(D) PET/CT scans of the patient are shown at (B) baseline before ACT, (C) during tumor regression on day +35 of ACT and (D) during tumor progression on day +84 of ACT. Red arrows indicate biopsied tumors and black arrows indicate progressing lesions at day +35. (E)–(G) Immunohistochemistry of the patient’s tumor for expression of S100, MART-1, CD8, gp100 and tyrosinase at (E) baseline before ACT, (F) during tumor regression and (G) during tumor progression. A heterogeneous, multifocal loss of MART-1 and gp100 is seen during tumor regression, with complete loss of these melanocytic antigens at the time of tumor progression.

Figure 2

Loss of melanocytic antigens is associated with increased NGFR expression and T cell infiltration. (A)–(C) Immunohistochemistry of the patient’s tumor for S100, MART-1, CD8 and NGFR (CD271) at (A) baseline before ACT, (B) during tumor regression and (C) during tumor progression. The loss of MART-1 is correlated with higher NGFR expression and CD8 T cell infiltration in both regressing and progressing tumors, thus suggesting inflammation-induced tumor dedifferentiation of these tumors.

Figure 3

F5 TCR T cells induce dedifferentiation of human melanoma cell lines. (A) M397 cells were left untreated, or cultured for three days with conditioned media obtained from co-culture of either untransduced or F5 TCR T cells with M397 cells. Flow cytometry was subsequently performed for surface expression of MART-1 and NGFR. (B) Untransduced or F5 TCR T cells were co-cultured with M397 cells for six hours. Flow cytometry was subsequently performed with intracellular staining for TNFα.

Figure 4

TNFα treatment induces a reversible dedifferentiation of several BRAF^V600 mutant melanoma cell lines. (A) M229 and M262 BRAF^V600 mutant cell lines were treated with DMSO or TNFα (1000U/mL) for 3 days. The surface expression of MART-1 and NGFR at the end of this period was quantified using flow cytometry. (B) Normalized median fluorescence intensities of MART-1 and NGFR expression by flow cytometry in several BRAF^V600 mutant melanoma cell lines treated with DMSO or TNFα for 3 days. Red and blue boxes highlight increased and decreased expression with TNFα treatment, respectively. (C) GSEA analysis of differentially expressed genes between melanoma cell lines treated with TNFα or DMSO for 3 days. An enrichment of genes characteristic of TNFα signaling, EMT and neural crest stem cells was seen in TNFα treated cells, and an enrichment of genes involved in the MITF pathway was seen in DMSO treated cells. (D) M397 cells were left untreated or treated for 3 days with TNFα. Inflammatory media was then replaced with fresh culture media for 7 days, and cells were subsequently analyzed by flow cytometry for surface expression of MART-1 and NGFR.