ALK and RET Inhibitors Promote HLA Class I Antigen Presentation and Unmask New Antigens within the Tumor Immunopeptidome

Abstract

T-cell immunotherapies are often thwarted by the limited presentation of tumor-specific antigens abetted by the downregulation of human leukocyte antigen (HLA). We showed that drugs inhibiting ALK and RET produced dose-related increases in cell-surface HLA in tumor cells bearing these mutated kinases in vitro and in vivo, as well as elevated transcript and protein expression of HLA and other antigen-processing machinery. Subsequent analysis of HLA-presented peptides after ALK and RET inhibitor treatment identified large changes in the immunopeptidome with the appearance of hundreds of new antigens, including T-cell epitopes associated with impaired peptide processing (TEIPP) peptides. ALK inhibition additionally decreased PD-L1 levels by 75%. Therefore, these oncogenes may enhance cancer formation by allowing tumors to evade the immune system by downregulating HLA expression. Altogether, RET and ALK inhibitors could enhance T-cell-based immunotherapies by upregulating HLA, decreasing checkpoint blockade ligands, and revealing new, immunogenic, cancer-associated antigens.

Figure 1:. ALK inhibition decreased pERK levels and increased surface HLA levels in ALK mutated cell lines.

(A) Karpas 299 cells were treated with increasing concentrations of crizotinib for 3hrs and pERK and ERK (loading control) were measured by western blot. (B) After 72hrs of crizotinib treatment, flow cytometry was used to measure cell surface HLA-A, B, C on Karpas 299 cells. Similarly, SUDHL-1 cells were treated with crizotinib and (C) pERK and ERK and (D) cell surface HLA molecules were measured. Western blot for ERK and pERK on (E) Karpas 299 cells and (F) SUDHL-1 cells treated with the second-generation ALK inhibitor, ceritinib, for 3hrs. Flow cytometry analysis of HLA-A, B, C expression in (G) Karpas 299 cells and (H) SUDHL-1 cells treated with ceritinib for 72hrs. W6/32-APC antibody was used to measure HLA-A,B,C.

Figure 2:. RET inhibition in TPC1 cells led to decreased pERK levels and increased surface expression of HLA.

TPC1 cells, a papillary thyroid carcinoma line with a RET/PTC1 rearrangement, were treated with the RET inhibitor, AST487. (A) After 72hrs, cell surface HLA-A*02 was measured through flow cytometry. (B) pERK and ERK (loading control) were measured at 24hrs by western blot. Similar results were observed with two other RET inhibitors: BLU6864 and cabozantinib. (C) Cell surface HLA expression and (D) pERK and ERK expression after BLU6864 treatment. (E) Cell surface HLA expression and (F) pERK and ERK expression after Cabozantinib treatment. BB7-APC antibody was used to measure HLA-A*02.

Figure 3:. The regulation of HLA increase was at the transcript level.

(A) Representative western blots probing for HLA-A, beta-2-microglobulin (B2M), and GAPDH (loading control) in TPC1 cells at 72hrs after RET inhibitor treatments. (B) HLA and antigen processing machinery (TAP1, TAP2 and Beta-2 microglobulin) transcript levels measured by qPCR at 48hrs after RET inhibitor treatment. (C) Western blots for HLA-A, beta-2-microglobulin (B2M), and GAPDH (loading control) and (D) RNA levels of for HLA-A, beta-2-microglobulin (B2M), and TAP-1 and TAP-2 in Karpas 299 cells and SUDHL-1 cells after ceritinib treatment. qPCR experiments were in performed in technical triplicate.

Figure 4:. Increased tumoral surface HLA expression and decreased tumoral PD-L1 expression in vivo during ALK inhibition.

(A) TPC1 cells were subcutaneously injected into NRG mice and harvested after 7 days of AST487 treatment or vehicle treatment (n=5). Cell surface HLA-A*02:01 and HLA-A, B, C were measured (with BB7 and W6/32 respectively). (B) PD-L1 levels were measured after RET inhibition. (C, D) Karpas 299 cells were subcutaneously injected into NSG mice, treated with alectinib, and harvested (n=5). HLA-A, B, C and PD-L1 levels were measured by flow cytometry.

Figure 5:. Mass spectrometry of eluted HLA class I presented peptides show a change in peptide number and repertoire after RET inhibition.

(A) HLA bound peptides from lysate of TPC1 cells treated with DMSO (control), 10nM AST487, or 100nM cabozantinib were analyzed by mass spectrometry (n=3). Only peptides found in all three separate runs were counted. Each circle encompasses the unique peptides identified after treatment; overlaps of circles show presence of each peptide in 2 or more groups. 458 and 492 new peptides appeared in each of the two RET inhibitor groups, respectively. (B) IFN-gamma ELISpot data for T cells stimulated with new arising peptides after RET inhibitor treatment (TLSGHSQEV, VYSLIKNKI, SYNEHWNYL, ALSGLAVRL). A representative figure is shown of two. PHA was used as a positive control. An irrelevant peptide (GRKPPLLKK) and CD14+ cells were used as negative controls.

Figure 6:. Unmasked antigen led to lysis of TPC1 cells by a TCR mimic antibody.

(A) ESK1, a TCR mimic antibody, was fluorescently labeled to probe binding after 72 hours RET inhibitor treatment by flow cytometry. (B) Chromium-51 labeled TPC1 cells were incubated with ESK1 and human PBMCs for 5hrs at 37°C and percent specific lysis was calculated for DMSO (control) and AST487 treated groups.

Figure 7:. Simplified schema of signaling pathway for HLA upregulation.

MEK, ALK or RET positively regulate the output of the MAPK pathway, which in turn downregulates STAT1, which leads to reduced HLA. Inhibitors of these kinases, reverse the process.