Combination JAK Inhibition and Immune Checkpoint Blockade for the Treatment of 9p Amplified Lymphomas

Background

Amplification of 9p24.1 (9p) upregulates PDL-1/2 and JAK2, leading to an immune-privileged tumor microenvironment (TME) and lymphomagenesis. The 9p amplicon is a key driver of disease in related lymphomas found in 100% of Hodgkin Lymphoma (HL), and 75% of Primary Mediastinal B-cell lymphoma (PMBCL). We hypothesize that if overexpression of PDL-1/2 and JAK/STAT leads to immune escape and unchecked proliferation, then dual targeting with ruxolitinib (RUX, JAK2 inhibitor) and pembrolizumab (PEM, PD-1 inhibitor) will target complementary pathways and enhance immunogenicity, inducing cell death.

Methods

The IC₅₀ values were established for RUX in four 9p positive (9p+) and negative (9p-) human lymphoma cell lines: K1106p (PMBCL, 9p+) 5.72 µM, L540 (HL, 9p+) 22 µM, Farage (PMBCL, 9p-) 29.46 µM, and U2940 (PMBCL, 9p-) 32.99 µM (Cell TiterGlo). To understand how RUX modulates markers of lymphomagenesis, western blot and flow cytometry was performed in all cell lines. Transcriptomic changes were evaluated by RNA-seq, presented as Log₂ fold change compared to vehicle control. To understand the effects of combination treatment on T-cell infiltration and function within the TME, PMBCL and HL organoids were developed and treated with RUX (0.5uM, 2.5uM, 5uM) and PEM (10ug/mL), and compared to monotherapy and vehicle controls. Following co-culture with donor CD8+ T-cells, organoids were stained with fluorescent calcein (live), ethidium (apoptotic), and CD3+ dyes and imaged on Leica SP8-DLC. Mean fluorescent values were calculated by Fiji. Reserved organoids were analyzed for T-cell function/activation and apoptosis (flow cytometry). Preliminary in vivo experiments were completed with partially HLA-matched humanized mice (HuMice) xenografted with K1106p.

Results

The effect with RUX treatment was most prominent in 9p+ cell lines: 9p+ K1106p (n=5) demonstrated increased markers of immunogenicity: CIITA 4.83-fold (SEM ± 1.15) and MHCII 2.67-fold (SEM ± 0.33). Tumor suppressor SOCS1 increased 3.33-fold (SEM ± 0.68), and MYC decreased 8.58-fold (SEM ± 4.66). The 9p+ L540 (n=5) paralleled these findings. In all cell lines, with and without 9p, RUX reduced expression of PDL-1/2 (p < 0.01) and MYC (p<0.05), (n=4, for each cell line). In K1106p treated with RUX, RNA-seq showed increased lymphocyte differentiation (p < 0.001), immune response activating pathways (p = 0.003), increased MHCII (p=0.001), decreased PDL-1/2 (-1.04, p < 0.009), and decreased MYC (-0.95, p < 0.001).

The 9p+ organoids (n=80) had the most significant decrease in live mean fluorescence with RUX+PEM compared to RUX alone (p=0.01) and to vehicle control (p=0.02). Intra-organoid T-cells (n=16) demonstrated increased CD3+ infiltration with RUX+PEM (p=0.01) compared to vehicle and monotherapy controls. There was significant decrease in intra-organoid PDL-1/2 ligands (p < 0.0001) with RUX treatment, suggesting a mechanism for synergy with RUX+PEM. Combination RUX+PEM demonstrated 43% reduction in total B-lymphocytes (p <0.001) with decreased live cells (p < 0.001) and increased dead cells (p=0.01) compared to vehicle and monotherapy controls (n=3). To confirm that RUX did not have a negative effect on T-cell viability and function, RUX treated T-cells demonstrated no change in live (p=0.71), dead (p=0.91), or apoptotic (p=0.6) T-cells, and no change in T-cell Ki-67 (p=0.78), granzyme-A (p=0.96), or granzyme-B (p=0.59), (n=3) compared to vehicle control. In K1106p HuMice, there was significant difference in tumor volume between control, and both treatment groups: RUX, and RUX+PEM (p=0.007, n=10), with stable weight (p=0.32) over 25 days of treatment. All drugs were safe and well tolerated. Doses have been optimized and there were no signs of graft vs. host disease in control or treated groups. Full in vivo studies will follow.

Conclusion

Altogether, these data suggest that RUX enhances immunogenicity and modulates lymphomagenesis primarily in 9p+ cell lines with increased CIITA, MHCII, and decreased MYC. In 9p+ organoids, combination treatment with RUX+PEM enhanced cell death via reduction in PDL-1/2 and increased T-cell infiltration. Deleterious effects of RUX on T-cell function were not observed. Full mouse model experiments are currently underway, laying the preclinical groundwork for a future clinical trial employing a multipronged approach for the treatment of 9p+ lymphomas.

*Funded by ASH RTAF.