JAK1 truncating mutations in gynecologic cancer define new role of cancer-associated protein tyrosine kinase aberrations

Abstract

Cancer-associated protein tyrosine kinase (PTK) mutations usually are gain-of-function (GOF) mutations that drive tumor growth and metastasis. We have found 50 JAK1 truncating mutations in 36 of 635 gynecologic tumors in the Total Cancer Care® (TCC®) tumor bank. Among cancer cell lines containing JAK1 truncating mutations in the Cancer Cell Line Encyclopedia databank, 68% are gynecologic cancer cells. Within JAK1 the K142, P430, and K860 frame-shift mutations were identified as hot spot mutation sites. Sanger sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed the JAK1 mutations and showed that these mutations are somatic. JAK1 mediates interferon (IFN)-γ-regulated tumor immune surveillance. Functional assays show that JAK1 deficient cancer cells are defective in IFN-γ-induced LMP2 and TAP1 expression, loss of which inhibits presentation of tumor antigens. These findings identify recurrent JAK1 truncating mutations that could contribute to tumor immune evasion in gynecologic cancers, especially in endometrial cancer.

Figure 1. JAK1 truncating mutations found in the TCC human tumors and CCLE cancer cells.

Schematic representation of JAK1 protein and the truncating mutation sites identified in the TCC tumors and CCLE cells. Vertical bars above JAK1 are proportional to the numbers of mutations found in the TTC and CCLE datasets. * indicates positions of nonsense mutations. Horizontal bars end at the position of stop codons.

Figure 2. Sanger DNA sequencing to examine JAK1 frame shift mutation at K860.

Genomic DNA of Hela cells (a), IGROV1 cells (c), an endometrial tumor (2T) or the matched normal endometrial tissue (2N) from the same patient (d) were prepared and sequenced across the K860 region. (b) CCLE igv_snapshot of the sequence alignment that identifies the K860fs mutation in IGROV1 cells.

Figure 3. JAK1 deficient cancer cells are defective in the IFN-γ-stimulated LMP2/TAP1 antigen-processing machinery pathway.

(a) Cell lysates from 6 cell lines were analyzed by immunoblotting with an anti-JAK1 antibody. Position of the 130-kDa full-length JAK1 is indicated. Other bands appear on the immunoblot are unknown non-specific immunoreactivities. (b–c) Cells were treated with IFNγ for the indicated time. Cell lysates were analyzed by immunoblotting for IFN-γ-induced pSTAT1, IRF1, LMP2, and TAP1.

Figure 4. Analysis of IFN-γ-induced LMP2- and TAP1-promoter activities.

(a) Comparison of IFN-γ-induced, IRF1-dependent LMP2 and TAP1-promoter activities in GYN cancer cell lines. The basal LMP2-Luc or TAP1-Luc activity, respectively, in Hela cells was set as 1 for comparison of Luc activities. Data were from two independent experiments performed in duplicate (n = 4). (b) JAK1 mutation cells were transfected with pCMV-JAK1 or the control vector and LMP2/TAP1-promoter reporter plasmids as in (a). IFN-γ-stimulated LMP2- and TAP1-promoter luciferase activities were assayed. *, p < 0.05 by the Mann-Whitney test. Differences in the basal LMP2- and TAP1-luciferase activities between cells transfected with pCMV-JAK1 and the control vector were p < 0.05 in all cell lines.

Figure 5. Effects of IFN-γ on cell proliferation/viability of GYN cancer cells.

(a) Cells were treated with IFN-γ for 4 days and viable cells were measured. Data were from two independent experiments performed in quadruple. *, p <0.05 by the Mann-Whitney test. (b) Cells were treated with IFN-γ for the indicated time and cell lysates were analyzed by immunoblotting with indicated antibodies.

Figure 6. Analysis of cell surface HLA molecules.

The amounts of HLA-ABC on the Hela, MFE296, and HEC1B cell surface were analyzed by flow cytometry after immunostaining with a PE-conjugated anti-HLA-ABC antibody as described in the Method. Data were from a representative experiment.