Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma

Abstract

Background: Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown.

Methods: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later.

Results: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I.

Conclusions: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).

Figure 1. Clinical Pattern of Acquired Resistance to Anti–Programmed Death 1 (PD-1) Therapy in Patient 1

In Panel A, computed tomographic images show a melanoma small-bowel metastasis at baseline, at the time of maximum response, and at the time of an in situ relapse after a year of minimal residual disease; the red dots in the graph on the right indicate these three time points. Immunohistochemical staining and multiplexed immunofluorescence analysis showed abundant CD8 T-cell infiltrates and programmed death ligand 1 (PD-L1) expression at baseline and again at the time of relapse at the tumor margin in Panels B and C, respectively. In the immunofluorescence images, red indicates PD-L1, yellow CD8 T cells, light blue the melanoma marker S100 (cytoplasmic staining), and dark blue the melanoma marker Sox10 (nuclear staining).

Figure 2. Acquired JAK1 Loss-of-Function Mutation at Relapse, with Accompanying Loss of Heterozygosity

In Panel A, a Circos plot of Patient 1 shows differences in whole-exome sequencing between the pre-pembrolizumab and post-relapse biopsies. The red circle highlights a new, high-allele-frequency, relapse-specific mutation in the gene encoding Janus kinase 1 (JAK1) in the context of chromosomal loss of heterozygosity (asterisk). Each wedge represents a chromosome. In the outer track (black background), each point represents a nonsynonymous mutation, with most detected in both biopsy samples (gray) rather than at relapse only (red) or baseline only (green). The y-axis position indicates the variant allele frequency (VAF) at relapse, unless baseline-specific. The middle and inner tracks show copy-number status for the baseline and relapse biopsy, respectively; dark green in the subtrack indicates loss of heterozygosity. In Panel B, Integrative Genomics Viewer (IGV) plots (top) show that the JAK1 Q503* nonsense mutation is relapse-specific, and the cBioPortal diagram (bottom) shows that the JAK1 mutation is upstream of the kinase domains.

Figure 3. Loss of Interferon Gamma–Induced Signaling and Gene-Expression Changes through Acquired JAK2 Mutation

In Panel A, Western blot analysis of lysates from cell lines M420 (Patient 2, baseline) and M464 (Patient 2, relapse) shows Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling events and downstream target induction after either 30 minutes (m) or 18 hours (h) of exposure to interferon (IFN) alfa, beta, or gamma (C indicates untreated control). Janus kinase 2 (JAK2) protein expression was absent in the relapse cell line (asterisk), and M464 failed to phosphorylate intermediate signaling components STAT1 and STAT3 or to up-regulate interferon-response targets TAP1, PD-L1, and major histocompatibility complex (MHC) class I after treatment specifically with interferon gamma (red box), as compared with intact signaling in M420 (blue box). There was no change in response to interferon alfa or beta. As shown in Panel B, a lack of response to interferon gamma exposure was also seen in surface staining for PD-L1 and MHC class I by flow cytometry. Each point represents an independent experiment, T bars represent standard deviations, and P values are for a two-way analysis of variance with Dunnett’s correction. MFI denotes mean fluorescent intensity, and NS not significant. Panel C shows log₂ RNA counts of expression for 790 immune-related genes on exposure to interferon gamma or vehicle control. The baseline cell line M420 (top) showed up-regulation of many interferon-stimulated genes (line represents an increase by a factor of 4), whereas the JAK2 mutated progression cell line M464 (bottom) lacked a similar response.

Figure 4. Loss of Interferon Gamma– Induced Growth Arrest through Acquired JAK Mutations

In Panel A, the M407 parental cell line as well as the M407 JAK1-knockout and JAK2-knockout sublines were recognized by NY-ESO-1–specific, HLA-A*02:01–restricted T cells, as assessed by interferon-γ production after 24 hours of in vitro coculture. M420 is negative for HLA-A*02:01 and served as a negative control. In Panel B, cell lines M420 and M407 showed growth inhibition in response to direct in vitro treatment with interferon alfa, beta, or gamma (left), whereas the JAK2-deficient counterpart M464 and the M407 JAK2 knockout were insensitive specifically to interferon gamma (middle). The M407 JAK1 knockout was insensitive to all three interferons (right). In Panel C, treatment with 2′3′-cGAMP (cyclic guanosine monophosphate–adenosine monophosphate), a direct cytosolic agonist of the stimulator of interferon genes (STING), was able to produce growth arrest in all cell lines, regardless of JAK2 status, yet had no effect in M407 with JAK1 knockout. Growth curves represent the percent change in the number of melanoma cells over time as measured by IncuCyte continuous live-cell imaging in one of three independent experiments. I bars in Panels A, B, and C indicate standard deviations for three replicate wells. Three asterisks indicate P<0.001 and two asterisks P<0.01 for the percent change in growth with the treatment shown at the 72-hour end point as compared with the untreated control, with Dunnett’s multiple-comparison correction applied in Panel B. NS denotes not significant.