Why Are Some Patients Unresponsive to Immunotherapy?

Immunotherapy is a promising treatment approach in the oncology landscape, but some patients are unable to respond to the therapy. In a study published in Cancer Discovery, investigators found that mutated JAK1 and JAK2 play a key role in primary resistance to anti-programmed death protein 1 (PD-1) therapy.

Loss-of-function mutations in JAK1/2 is associated with acquired resistance to PD-1 blockade in patients with advanced melanoma. The lack of interferon gamma responsiveness allows cancer cells to evade antitumor T cells, resulting in the loss of PD-L1 expression.

For the study, the investigators sought to determine the role of JAK1 and JAK2 disruption in primary resistance to PD-1 blockade therapy by performing a genetic analysis of tumors from patients with advanced melanoma and colon cancer who were unresponsive to PD-1 blockade therapy, despite having a high-mutational load.

The investigators performed whole-exome sequencing (WES) of 23 pretreatment biopsies obtained from patients with advanced melanoma treated with pembrolizumab (Keytruda), of whom, 14 had a tumor response by immune-related RECIST (irRECIST) criteria and 9 without a response.

The authors noted that although the mean mutational load was higher in responders compared with nonresponders, some patients with a tumor response had a low mutational load and some patients without a tumor response had a high mutational load.

The investigators examined whether loss-of-function mutations in interferon receptor signaling molecules were present in tumors with a relatively high mutational load that was unresponsive to therapy, according to the study.

Of the 9 nonresponders, only 1 with the highest mutational load had a somatic P429S missense mutation in the src-homology (SH2) domain of JAK1. According to the study, none of the tumors derived from the other 22 patients had homozygous loss-of-function mutations or deletions in the interferon receptor pathway. Instead the other JAK2 mutations found in the biopsies of responders had low variant allele frequency (VAF), and were likely heterozygous.

“As expected, tumors from patients who responded had a higher density of CD8 cells and PD-L1 in the center and invasive tumor margin,” the authors wrote. “In contrast, the baseline biopsy from patients #15 with a high mutation load but with JAK1 P429S missense mutation had undetectable CD8 infiltrates, PD-1, and PD-L1 expression.”

To characterize the interferon response of M431 with high mutational load and no response to therapy, the investigators optimized flow cytometry conditions in selected human melanoma cell lines. The results of the study showed that PD-L1 expression increased less than 1.5-fold interferon gamma exposure in M431 compared with 5.1-fold in M438, a cell line established from patient number 8 and used as a positive control in the same series.

Additionally, phosphorylated STAT1 (pSTAT1) was induced at 30 minutes in M431, however, the signal dissipated at 18 hours. This was faster than what was observed in cell lines with more durable responses to interferon gamma leading to PD-L1 regulation, according to the study.

Next, the investigators screened a panel of 48 human melanoma cell lines for absolute absence of PD-L1 induction by type 1 or type 2 interferons. Among the 3 interferons, the investigators found that interferon gamma most potently induced PD-L1 expression.

Signaling was analyzed in response to interferon alpha, beta, and gamma, in the 2 cell lines M368 and M395.The results showed that M368—–which harbored the JAK2 loss-of-function mutation––maintained signaling in response to interferon alpha and beta, but did not respond to interferon gamma. This results in M368 upregulating PD-L1 when exposed to interferon alpha and beta but not to interferon gamma.

M395 harbored the JAK1 loss-of-function mutation and did not respond to downstream signaling to interferon alpha, beta, or gamma, and equally did not upregulate PD-L1 in response to any of the cytokines, according to the authors.

“To assess a causal relationship between loss of adaptive PD-L1 expression and loss-of-function JAK mutations, we transduced the M395 and M431 cell lines with a lentivirus vector expressing JAK1 wild-type,” the authors wrote. “Reintroduction of wild-type JAK1 rescued PD-L1 expression in M395 cells, which exhibited a 4-fold increase in PD-L1 surface expression after interferon exposure.”

After 18-hour interferon gamma exposure for M431, the magnitude of change in PD-L1 expression was modest after reintroducing the JAK1 wild-type protein.

The authors noted that the difference between untransduced and JAK1 wild-type transduced M431 was more distinct when observed over a longer period of time.

To determine whether JAK1/2 loss-of-function mutation are present and related to PD-1 blockade therapy response in metastatic colon carcinoma, the investigators analyzed WES data from 16 biopsies of patients with colon cancer, many of whom had a high mutational load as a result of mismatch-repair deficiency.

One biopsy of a patient with a high mutational load with neither an objective response nor disease control with anti-PD-1 had a homozygous JAK1 W690 nonsense loss-of-function mutation found that no mutations in antigen presentation machinery were detected in the sample.

To determine the frequency of homozygous putative loss-of-function mutations in JAK1.2 in 905 cancer cell lines, the investigators analyzed data from the Cancer Cell Line Encyclopedia from cBioPortal. A homozygous mutation was defined as the VAF at 0.8 or greater.

Approximately 0.7% of cell lines had loss-of-function mutations that may predict lack of response to interferons. The highest frequency was found in endometrial cancers. None of the cell lines had POLE of POLD1 mutations, however, microsatellite instability and DNA-damage gene mutations were present in the JAK1/2 mutant cell lines, according to the authors.

“The frequency of JAK1/2 mutations across all cancers suggests that there is a fitness gain with loss of interferon responsiveness,” the authors wrote.

The investigators performed a genetic analysis of tumors from patients with melanoma and colon cancer who did not respond to PD-1 blockade therapy despite having a high-mutational load. They identified tumors with homozygous loss-of-function mutations in JAK1 and JAK2 and studies the functional effects of deficient interferon gamma receptor signaling that lead to a genetically mediated absence of PD-L1 expression upon interferon gamma exposure.

“A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes,” the authors wrote. “These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy.”

Currently, the investigators are examining animal models with JAK1 and JAK2 genetic mutations to develop new and improved combination therapies for patients who are unresponsive to anti-PD-1 treatment.