Ruxolitinib increases immunogenicity and primes the tumor microenvironment for immune checkpoint blockade in 9p24.1 amplified lymphomas Free

Amplification of 9p24.1 (9p+) upregulates PDL-1/2 and JAK2, leading to an immune-privileged tumor microenvironment (TME) and lymphomagenesis. This biology is relevant for lymphomas that harbor 9p+ or rely on JAK/STAT and immune escape pathways for growth, including Hodgkin Lymphoma (HL) and primary B-cell mediastinal lymphoma (PMBCL). Our previous data suggest that ruxolitinib (RUXO), a JAK1/2 inhibitor, enhances immunogenicity by increasing CIITA, MHCII, and decreasing c-MYC in cell lines, while also increasing T-cell infiltration in lymphoma organoids, priming the TME for PD-1 inhibition with pembrolizumab (PEMBRO). We hypothesize that RUXO disrupts the immune-privileged TME by increased T-cell activation and immune surveillance, thereby increasing the efficacy of PEMBRO in 9p+ and JAK/STAT mutated lymphomas to enhance tumor death when combined with PEMBRO.

To discern the effects of RUXO+PEMBRO on the peripheral immune milieu in patients, isolated peripheral blood mononuclear cells (PBMCs) were treated with vehicle, RUXO (1uM), PEMBRO (1mg/ml), or combination (COMBO); samples were analyzed by flow cytometry (HL = 7, PMBCL = 3). Compared to control, RUXO and COMBO increased peripheral immune surveillance via increased NK cells, decreased Tregs, and decreased immune suppressive myeloid derived suppressor cells (MDSCs) with all p<0.05; altogether suggesting an activated peripheral immune compartment.

To investigate effects of RUXO+PEMBRO on tumor growth and survival, immunized BALB/c mice were engrafted with the syngeneic A20 cell line (JAK3, STAT 2/4 mutated). Mice (n=72) were treated with vehicle control, RUXO (90mg/kg), PEMBRO (5mg/kg every 4 days), or COMBO until tumor volumes>2000 mm³. A significant decrease in tumor volume was observed with COMBO compared to vehicle and monotherapy controls. With median follow up of 30 days, Kaplan-Meier analysis showed significant differences in median survival for COMBO (not reached) compared to vehicle (17 days), RUXO (23 days), and PEMBRO (25 days) with p<0.0001. Treatment was well tolerated and there was no significant decrease in weight. To understand the impact of RUXO+PEMBRO on the peripheral immune milieu, n=6 mice were sacrificed from each cohort on day 7 for PBMC analysis by flow cytometry. Immune activating effects via decreased MDSCs and increased NK cells were observed with COMBO compared to control. RUXO and COMBO increased markers of activation (CD25+) while decreasing markers of exhaustion (LAG3 and PD-1) in both CD4+ and CD8+ T-cells (all p <0.05).

To understand the impact of RUXO+PEMBRO on the TME, humanized mice (huMice) partially HLA-matched to the human K1106p cell line (9p+) were treated with vehicle control, RUXO (30m/kg daily), PEMBRO (10mg/kg day 1, then 5mg/kg every 5 days), and COMBO; n=6 mice from each cohort were sacrificed at day 7 and analyzed by flow cytometry. PBMCs demonstrated increased markers of CD4+ and CD8+ T-cell activation (CD40L) with RUXO and COMBO. In tumor samples, RUXO and COMBO decreased PDL-1 expression. Tumor-infiltrating CD3+ T-cells were increased with RUXO and COMBO with increased markers of activation in CD4+ (CD40L) and CD8+ (CD40L, CD69) T-cells. A decreased CD4:CD8 ratio was observed in both PBMCs and tumor samples with RUXO and COMBO, shifting toward higher levels of activated CD8+ T cells.

huMice tumor samples were further analyzed by spatial Multiomics Single-Cell Imaging (CosMx™) for protein tissue microarray. After QC, normalization, and scaling, five batch-correction methods (CLR, SCTransform, Harmony, BANKSY, InsituType) were compared. Cohorts demonstrated distinct treatment-specific remodeling of the TME with increased clusters of activated CD3⁺ T-cells, dendritic cells, plasmablasts, macrophages, and antigen-presenting B-cells. Specifically, COMBO (n=9) increased tumor-infiltrating activated macrophages, NK cells, and activated CD3+ T-cells compared to control (n=7).

RUXO induces a shift toward an activated immune milieu, priming the 9+ TME for PEMBRO. RUXO+PEMBRO leads to synergistic anti-tumor activity, modulates the TME via increased infiltration of cytotoxic immune cells, and promotes a shift toward an activated CD8⁺ T cell-dominant TME. These data underscore the potential of JAK/PD-1 axis co-targeting to overcome immune evasion and enhance immunotherapeutic efficacy, leading to the development of an investigator-initiated trial in 9p+ and JAK/STAT mutated lymphomas.