Selective JAK2 inhibition specifically decreases Hodgkin lymphoma and mediastinal large B-cell lymphoma growth in vitro and in vivo

Abstract

Purpose: Classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (MLBCL) share similar histologic, clinical, and genetic features. In recent studies, we found that disease-specific chromosome 9p24.1/JAK2 amplification increased JAK2 expression and activity in both cHL and MLBCL. This prompted us to assess the activity of a clinical grade JAK2 selective inhibitor, fedratinib (SAR302503/TG101348), in in vitro and in vivo model systems of cHL and MLBCL with defined JAK2 copy numbers.

Experimental design: We used functional and immunohistochemical analyses to investigate the preclinical activity of fedratinib and associated biomarkers in cell lines and murine xenograft models of cHL and MLBCL with known 9p24.1/JAK2 copy number.

Results: Chemical JAK2 inhibition decreased the cellular proliferation of cHL and MLBCL cell lines and induced their apoptosis. There was an inverse correlation between 9p24.1/JAK2 copy number and the EC50 of fedratinib. Chemical JAK2 inhibition decreased phosphorylation of JAK2, STAT1, STAT3, and STAT6 and reduced the expression of additional downstream targets, including PD-L1, in a copy number-dependent manner. In murine xenograft models of cHL and MLBCL with 9p24.1/JAK2 amplification, chemical JAK2 inhibition significantly decreased JAK2/STAT signaling and tumor growth and prolonged survival. In in vitro and in vivo studies, pSTAT3 was an excellent biomarker of baseline JAK2 activity and the efficacy of chemical JAK2 inhibition.

Conclusions: In in vitro and in vivo analyses, cHL and MLBCL with 9p24.1/JAK2 copy gain are sensitive to chemical JAK2 inhibition suggesting that clinical evaluation of JAK2 blockade is warranted.

Figure 1. Fedratinib inhibits proliferation and induces apoptosis in cHL and MLBCL cell lines

(A) Cellular proliferation of cHL cell lines (L428, KMH2, L1236, SUPHD1 and HDLM2) and the MLBCL cell line (K1106P), following treatment with vehicle or fedratinib at indicated concentration for 48 hours. For each cell line, the previously reported 9p24.1/JAK2 copy numbers (7) are indicated in parenthesis. At a given dose of the JAK2 inhibitor (1.25 μM), a Kruskal-Wallis test was performed to assess the association between the ranked values of inhibition and copy number gain (p = .009, cHL and MLBCL cell lines; p = .019, cHL cell lines). (B) Apoptosis (annexin V⁺/DAPI⁻ plus annexin V⁺/DAPI⁺) of the cHL and MLBCL cell lines following 48 hours of treatment with vehicle or fedratinib. Data in (A) and (B) are representative of three independent experiments.

Figure 2. Fedratinib inhibits JAK2/STAT signaling pathway in cHL and MLBCL cell lines

(A) Western analysis of pJAK2 and the downstream pSTATs following treatment with vehicle or the indicated concentrations of fedratinib for 24 hours. Total JAK2 and GAPDH are similarly analyzed. (B) Analysis of pJAK2 and the downstream pSTATs following vehicle or fedratinib (2.5 μM) treatment for 2 – 24 hours. Data in both (A) and (B) are representative of three independent experiments.

Figure 3. Immunohistochemical signatures of chemical JAK2 inhibition in cHL and MLBCL cell lines

Immunohistochemical analyses of pJAK2, pSTAT1 and pSTAT3 expression in the HDLM2 cHL cell line (A) and the K1106P MLBCL cell line (B) following treatment with vehicle or fedratinib (2.5 μM) for 24 hours. (C) Immunohistochemical analysis of pSTAT3 in a primary cHL with known 9p24.1/JAK2 amplification (original magnification 400x, Scale bar = 100 μm).

Figure 4. Chemical JAK2 inhibition decreases PD-L1 expression in cHL and MLBCL

(A) RT-qPCR analysis of PD-L1 transcript abundance in cell lines treated with vehicle or fedratinib for 24 hours. (B) Flow cytometric analysis of cell surface PD-L1 expression in cells treated with vehicle or fedratinib for 48 hours. Data in both (A) and (B) are representative of three independent experiments.

Figure 5. Chemical JAK2 inhibition modulates c-MYC and PIM1 expression in cHL and MLBCL cell lines

Western analysis of c-MYC and PIM1 protein levels in cHL and MLBCL cell lines treated with vehicle or fedratinib at the indicated concentration for 24 hours. Data are representative of three independent experiments.

Figure 6. Chemical JAK2 blockage inhibits cHL and MLBCL tumor growth in murine xenograft models

Luciferized Karpas 1106P cells were xenotransplanted into NOD SCID IL2rγnull (NSG) mice and monitored by bioluminescence imaging. (A) Immunohistochemical analysis of bone marrow from Karpas 1106P xenograft mice following 5 days treatment with vehicle or fedratinib (4 mice in each group). Infiltrating tumor cells were stained with anti-CD20 and anti-pSTAT3 antibodies, and representative staining is shown at 400x magnification, scale bar=200μm. Higher magnification (900x) is shown in insert at upper left corner. (B) Bioluminescence of vehicle- or fedratinib-treated Karpas 1106P mice (10 mice in each group). Error bars show the SEM. P values obtained with student t-test. (C) Survival of Karpas 1106P mice treated with vehicle or fedratinib (10 mice in each group). P values obtained with a log-rank (Mantel-Cox) test. Luciferized mCherry+ HDLM2 cells were inoculated subcutaneously into NSG mice. (D) Immunohistochemical analysis of pSTAT3 expression in a representative tumor mass from HDLM2 xenograft mice following 5 days treatment with vehicle or fedratinib (4 mice in each group). Representative staining is shown at 200x magnification, scale bar=200μm. Higher magnification (500x) is shown in insert at upper left corner. (E) Tumor volume in HDLM2 mice (10 mice in each group) measured by calipers. Error bars show the SEM. (F) Single cell suspensions were prepared from HDLM2 tumor masses at the end of treatment with vehicle or fedratinib (6 mice in vehicle group and 10 mice in fedratinib group) and residual viable tumor cells were analyzed for pSTAT3 expression by intracellular phosphoflow cytometry. Left) Representative pSTAT3 expression in vehicle- and fedratinib-treated tumor cells. Right) Comparison of pSTAT3 expression in the vehicle- and fedratinib-treated cohorts. Fold change was calculated by comparing median fluorescence intensity values for pSTAT3 over isotype control for each sample. P values in (E) and (F) obtained with student t-tests.