The Small Molecule JAK1/JAK2 Inhibitor INCB16562 Shows Single Agent Activity and Strongly Synergizes with Bcl-2 Inhibitors in Lymphoma

Abstract 2731

Constitutive activation of Janus Kinases has been shown to be an important mechanism supporting human lymphomagenesis. Aberrantly activated JAK/STAT signaling has been described in Hodgkin lymphoma (HL), Anaplastic Large T-cell lymphoma and Diffuse large B cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, making it an attractive target for therapy. In this study we report the activity profile of the JAK2 inhibitor INCB16562 in a panel of lymphoma cell lines (n=17) including Hodgkin (HL), Mantle Cell (MCL), Anaplastic Large T-cell (ALCL), and DLBCL. JAK-STAT pathway was found to be constitutively active (baseline p-STAT3 Tyr 705 detected by western immunoblotting) in the HL (HDLM-2, L.-428 and L-540), ALCL (SUP-M2, KARPAS-299, SUDHL-1) and the ABC derived DLBCL cell lines (HBL-1, U2932, TMD8, OCI-LY-3). In contrast, no activation of JAK-STAT was observed in the HL cell line KM-H2, in the MCL cell lines (Mino, Jeko-1, SP-53) and in the GCB-derived cell lines (SUDHL-4, SUDHL-6, BJAB). To assess the effect of INCB16562 on cell proliferation, cells were first incubated with increasing concentrations of INC16562 (from 0.1 to 10 μM) for 24, 48 and 72 hours (hrs) and cell viability was evaluated by MTS assay. The IC50 values at 48 hrs ranged from 1 to 9 μM. The ABC derived DLBCL cell line TMD8 showed the highest sensitivity, with a decrease in cell viability close to 50% following incubation with INCB16562 1μM for 24 hours. Of note, INCB16562 demostrated activity also in primary DLBCL cells. pSTAT3 negative cell lines were resistant to treatment (IC50 from 4.8 to 6.5 μM). However pretreatment level of pJAKs and pSTATs did not predict sensitivity to INCB16562. In fact inhibition of STAT3 phosphorylation alone was not sufficient to predict sensitivity to the drug in pSTAT3 expressing cells. Simultaneous inhibition of STAT3 and ERK phosphorylation was observed by western immunoblotting in the sensitive cell lines (TMD8, HBL-1, SUP-M2, KARPAS 299, SUDHL-1) (IC50 from 1 to 3 μM), whereas in the resistant cell lines (U2932, OCI-LY3, HD-LM2, L-540, L-428) a paradoxical hyperactivation of pERK was observed after incubation with INCB16562 (IC50 from 5–9 μM). Since it has been reported that sensitivity to JAK inhibition is associated to downregulation of bcl-2 family genes such as Bcl-xL and MCL-1, we investigated the baseline expression of these bcl-2 family members in our cell lines by western blot. Bcl-xL and MCL-1 were expressed at various levels, but there was no correlation between levels of baseline expression and outcome. Both Bcl-xL and MCL-1 were dowregulated in the sensitive cell lines following treatment with INCB16562. Furthermore The bcl-2 inhibitor ABT-737 synergistically enhanced the effect of INCB16562 particularly in HL cell lines (HDLM-2 and L-540) and DLBCL cells lines (HBL-1, TMD8, SUDHL-4). These data demonstrate that INCB16562 has a promising therapeutic value in lymphoma, and provide a rationale for combining INCB16562 with bcl-2 family inhibitors.