T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin lymphoma (T-NHL), are a biol. and clin. heterogeneous group of rare and aggressive neoplastic disorders that result from clonal expansion of lymphocytes committed to the T-cell lineage.Although the prognoses for T-cell malignancies have improved in recent years due to intensified combination chemotherapies, the outcome of patients with resistant or relapsed disease remains poor.Therefore, current research efforts are focused on more effective and less toxic targeted therapies, which will require an improved understanding of the mol. events underlying these malignancies.Janus kinase 3 (JAK3) is constitutively activated in a number of T-cell malignancies and has emerged as a mol. target for cancer therapy.3, 4, 5 JAK3 belongs to a family of cytoplasmic non-receptor tyrosine kinases (JAK1, JAK2, JAK3 and TYK2) that mediate signals initiated by cytokine and growth factor receptors.6 Although JAK1, JAK2 and TYK2 are ubiquitously expressed, JAK3 is primarily confined to the hematopoietic lineage, where it has a vital role in lymphoid cell development and homeostasis.7 Its restricted expression and critical function in lymphoid cells has attracted significant attention in recent years as a therapeutic target for immune-mediated diseases;8 however, achieving JAK3 selectivity has remained a significant challenge.9 The first-in-class JAK3 inhibitor, tofacitinib, has been hindered by undesirable pan-JAK inhibitory side effects that include anemia and neutropenia, presumably related to JAK2 inhibition and interference with cytokines such as erythropoietin and colony-stimulating factors.10, 11 Thus, although efficacy of a next-generation JAK3 inhibitor is paramount, drug selectivity to JAK3 over JAK2 may determine its clin. usefulness.By a series of structure-activity relationship studies based on the chem. scaffold of the established JAK3 inhibitor NC1153,12 we identified a novel, stable, selective and efficacious inhibitor of JAK3 that was designated EP009 (Figure 1a, Supplementary Figure S1).In vitro kinase assays revealed EP009 to be a low micromolar inhibitor of JAK3 (Figure 1b) with limited off-target effects against a panel of 92 human kinases (Supplementary Table S1).The interleukin (IL)-2-dependent T-cell line Kit225 and the IL-3-dependent pro-B-cell line BaF/3 were utilized to test EP009 specificity for JAK3 vs. JAK2, resp.In Kit225 cells, EP009 reduced IL-2-mediated JAK3 tyrosine phosphorylation with a cellular IC50 between 10 and 20 μ (Figure 1c, upper panel).In contrast, EP009 had no detectable effect on IL-3-induced JAK2 tyrosine phosphorylation in BaF/3 cells up to 50 μ (Figure 1c, lower panel).In addition, EP009 treatment reduced Kit225 cell viability with a LD50 of 5.0 μ at 72 h, whereas no effect on BaF/3 cell viability was detected (Figure 1d).Structure-activity relationship studies revealed that deletion of the C12 methylene or substitution at the C2 hydroxymethyl groups of the cyclododecanone ring significantly reduced the efficacy of EP009 (Supplementary Figure S2).Under the same exptl. conditions, JAK inhibitors such as tofacitinib (CP-690,550) and ruxolitinib (INCB-18424) reduced both Kit225 and BaF/3 cell viability (Supplementary Figure S3).Thus, although the IC50 values suggest that EP009 is not as potent as other JAK inhibitors, the degree of kinase selectivity differentiates it from what is typically obtained from an ATP-competitive inhibitor.Ongoing studies seek to delineate the underlying mechanism of JAK3 inhibition by EP009 through the application of biochem., biophys. and structural studies.To assess possible off-target effects of EP009 on renal, hepatic and peripheral lymphocytic systems, we examined its impact on human cells including the kidney cell line HEK293, liver cell line HEPG2 and primary naive PBMCs, resp.Cell treatment with 10 μ EP009 for 72 h resulted in no detectable loss of cell viability (Figure 1e), suggesting a favorable in vitro cytotoxicity profile for EP009.Thus, preclin. investigations into the efficacy of EP009 against T-cell malignancies that display constitutively activated JAK3 were initiated using representative T-NHL anaplastic large cell lymphoma (ALCL) cell lines (Supplementary Figure S4).13 EP009 treatment led to a concentration-dependent reduction in viability of NPM-ALK-pos. SU-DHL-1 and SUP-M2 ALCL cells with an LD50 of 5 μ at 72 h (Figure 1f).In contrast, EP009 had no effect on cell viability of the EML4-ALK-pos., but JAK3-neg., non-small cell lung cancer cell line H2228, further supporting the selectivity of EP009 toward JAK3 and its functional role in T-NHL ALCL.Similarly, we examined the effects of EP009 on the viability of human cutaneous T-cell lymphoma cell lines such as H9 and HH.EP009 reduced the viability of HH cells with a LD50 between 2.5 and 5 μ at 72 h; however, identical concentrations had limited effects on H9 cells (Figure 1g).Western blot anal. of total cell lysates revealed that although JAK3 protein expression was readily detectable in the EP009-sensitive HH cells, its presence in the EP009-refractory H9 cells was below the level of detection (Figure 1g, upper panel).These data further support the hypothesis that EP009 selectively disrupts JAK3-dependent tumor cell viability.The in vitro results using tumor T-cell lines prompted us to assess the ex vivo efficacy of EP009 against primary cultures of leukemia patient tumor cells.The cytotoxic effect of EP009 was evaluated in PBMCs obtained from two patients diagnosed with relapsed T-ALL (Supplementary Table S2).EP009 treatment led to a concentration-dependent reduction in viability of T-ALL-P1 cells with an LD50 between 2.5 and 5 μ at 72 h (Figure 1h).In contrast, EP009 had no effect on T-ALL-P2 cell viability at 72 h.Western blot and densitometry anal. of total cell lysates revealed 80fx less JAK3 protein expression in the T-ALL-P2 cells, suggesting a correlation between the level of JAK3 protein expression and EP009 sensitivity.Taken together, these findings indicate that EP009 represents a viable therapeutic strategy for the treatment of JAK3-dependent T-cell malignancies.To further explore the functional consequences and in vivo activity of EP009-mediated JAK3 inhibition, we utilized the human T-NHL ALCL model system.Constitutive tyrosine phosphorylation and activation of JAK3/STAT3 in SU-DHL-1 cells was inhibited by EP009 in a dose-dependent manner (Figures 2a and b).The decrease in JAK3/STAT3 activation was associated with loss of cell viability that was attributed to the induction of apoptosis as confirmed by an increased caspase-3 activation and PARP cleavage (Supplementary Figures S5a and 5b).In addition, EP009 cytotoxicity correlated with the activation of JNK1/2, but not p38, mediated stress signaling and inactivation of p70 S6 kinase-mediated growth and survival signaling in SU-DHL-1 (Supplementary Figures S5c, 5d and 5e).These findings highlight the importance of stress generation through JAK3 inhibition that promotes apoptosis in T-NHL cells.To assess the clin. potential of EP009, its bioavailability was determined following the oral administration (200 mg/kg dosing) to Sprague Dawley rats.Pharmacokinetic anal. showed rapid bioavailability with peak blood concentrations (∼6 μ) at 30 min and was detectable up to 8 h (Figure 2c).Thus, tumor xenografts should be exposed to inhibitory concentrations of EP009 previously observed in the cell-based studies.Whether repetitive dosing results in greater tissue accumulation and EP009 concentrations remains to be determinedThe therapeutic efficacy of EP009 was further evaluated using a SCID/NOD murine xenograft model of human T-NHL ALCL.Treatment of mice bearing SU-DHL-1 tumors with EP009 given orally at 100 or 200 mg/kg resulted in significant tumor inhibition (>50fx reduction; P<0.01) vs. placebo control groups (Figure 2d).Tumor responses were evident 3 wk post EP009 treatment, suggesting greater tissue accumulation is required for inhibiting tumor cell growth.However, once initiated, tumor responses were maintained for the duration of the treatment.Consistent with the in vitro results, EP009 treatment was associated with reduced levels of Tyr705-phosphorylated STAT3 (P<0.01) compared with vehicle-treated control tumors (Figures 2e and f).Considering the effects of EP009 on ALCL cells in vitro and in vivo, these results suggest that constitutive activation of JAK3 represents a viable therapeutic target to treat malignant T-cell lymphoma.Indeed, supporting evidence indicates that JAK3 is a secondary oncogenic driver in ALCL that is induced by autocrine cytokine signaling mechanisms via IL-914 and IL-21.15 Therefore, EP009-mediated inhibition of JAK3 would serve as a logical therapeutic strategy for intervention in hematopoietic malignancies in primary response to JAK3 activating alleles and secondary responses where JAK3 acts as an oncogenic driver, such as ALK-pos. T-NHL ALCL.In conclusion, EP009 is a selective and orally active JAK3 inhibitor that displays a favorable cytotoxicity profile with therapeutic efficacy against JAK3-driven tumor T-cells in vitro, in vivo and ex vivo.Current cancer drug development is focused on targeted therapies that selectively uncouple cell signaling pathways required for tumor cell growth and survival.The data generated and described in this study support the role of JAK3 as a viable and relevant mol. target in the treatment of T-cell malignancies, where compounds such as EP009 can be effective anti-neoplastic agents.
