The JAK Inhibitor Pacritinib Suppresses the Checkpoint Proteins Pvrig and Tigit Expression on T Cells in Multiple Myeloma Patients

Introduction: Immunotherapies blocking immune checkpoints have been effective in cancer treatment, but these drugs have significant toxicities. Previously, we have shown the JAK1/2 inhibitor ruxolitinib has beneficial effects for treating multiple myeloma (MM) patients. We have also found that it reduces expression of two immune checkpoint proteins, PD-L1 and B7-H3, in MM bone marrow (BM). Pacritinib is another JAK inhibitor that specifically targets JAK2, but not JAK1. We hypothesized pacritinib may inhibit other immune checkpoints in MM. The immune checkpoint proteins TIGIT and PVRIG have been shown to be over-expressed in MM BM, and we evaluated whether pacritinib could reduce expression of these checkpoint inhibitors and the effect of this JAK inhibitor on MM growth.

Methods: The human MM cell lines U266, RPMI8226 and MM1s were obtained from ATCC. Mononuclear cells (MCs) were isolated from MM patients' BM aspirates. Cell viability was quantified using the MTS cell proliferation assay. We determined the IC curves for the MM cell lines RPMI8226, U266, and MM1S and fresh BMMCs from the MM patients using this JAK inhibitor and other anti-MM drugs. Specifically, we examined the anti-proliferative effects of pacritinib alone and in combination with dexamethasone, pomalidomide or bortezomib using these 3 MM cell lines and 1 patient's fresh MM BMMCs. In addition, 5 fresh MMBMMCs (3 patients with progressive disease [PD] and 2 patients in complete remission [CR]) were treated with pacritinib alone for 48 hours in vitro, followed by flow cytometric analysis to determine TIGIT and PVRIG expression using an Attune flow cytometer.

Results: MM tumor cells from the 3 human myeloma cell lines were exposed to increasing concentrations of pacritinib from 0.1 to 100 µM. The results of anti-MM effects on cell viability showed that pacritinib alone inhibited MM tumor cell proliferation in all three MM cell lines (RPMI8226, U266 and MM1S) in a concentration dependent manner. We also found the viability in one PD patient was significantly decreased when pacritinib was combined with dexamethasone or bortezomib compared to these drugs alone, but there was no effect when this JAK inhibitor was combined with pomalidomide.

Next, flow cytometric analysis showed PVRIG and TIGIT expression in MM BMMCs were significantly decreased after pacritinib treatment in vitro for 48 hours in both PD and CR patients, demonstrating that pacritinib inhibited PVRIG and TIGIT in MMBM. Specifically, pacritinib reduced the expression of both PVRIG and TIGIT on CD8+ T cells. Thus, blockade of PVRIG and TIGIT on CD8+ T cells with pacritinib may restore T cell anti-tumor activity.

Summary: We demonstrated that pacritinib can inhibit expression of PVRIG and TIGIT in MM BM, and specifically blocks PVRIG and TIGIT expression on CD8+ T cells. We also show the anti-MM effects of pacritinib both alone and in combination with other anti-MM drugs. Pacritinib can be potentially beneficial for treating myeloma patients due to its inhibitory role on PVRIG and TIGIT expression which may enhance tumor cell killing by CD8+ T cells.

No relevant conflicts of interest to declare.