Dual Inhibition of JAK/STAT and MAPK Pathways Results in Synergistic Cell Killing of JAK-Mutated Pediatric Acute Lymphoblastic Leukemia

Abstract 3562

Relapsed/refractory pediatric acute lymphoblastic leukemia (ALL) remains a continuing challenge to treat with currently available therapies, and new treatments are urgently required for the management of these high-risk cases. Activating mutations in the pseudokinase or kinase domains of Janus kinases (JAKs) 1, 2 and 3 are present in approximately 10% of high-risk pediatric ALL and are associated with high expression of cytokine receptor-like factor 2 (CRLF2) and poor outcome. These mutations can lead to continuous activation of JAKs, resulting in constitutive activation by phosphorylation of downstream signaling, including the signal transducer and activator of transcription (STAT), AKT, and mitogen-activated protein kinase (MAPK) pathways. The availability of specific JAK inhibitors, developed primarily for the treatment of JAK-mutated myeloproliferative diseases (MPDs), represents an opportunity to improve the treatment options for JAK-mutated pediatric ALL. AZD1480, a potent ATP-competitive small-molecule JAK2 inhibitor that also exhibits inhibitory activity against JAK1, is in solid tumor clinical trials. The purpose of this study was to gain a greater understanding of a potential role for AZD1480 in the treatment of JAK-mutated pediatric ALL either as a single agent or in rational drug combinations, using a preclinical model of xenografts established in immune-deficient mice from direct patient explants. As part of the Pediatric Preclinical Testing Program (PPTP) we previously showed that AZD1480 administered at 10 mg/kg twice daily × 5 then at 15mg/kg once daily × 2 via oral gavage for an intended three weeks significantly delayed the progression of only one in five JAK-mutated xenografts, with no tumor regressions observed. We now show that the relative insensitivity of JAK-mutated ALL xenografts to AZD1480 is a cell-intrinsic phenomenon, since 6/7 JAK1- or JAK2-mutated xenografts exhibited ex vivo IC50 values >2 μM following 72 h drug exposures, as assessed by mitochondrial function cell viability (MTT) assay. In order to gain a greater understanding of the underlying mechanisms for the lack of AZD1480 single-agent efficacy against JAK-mutated xenografts we analyzed intracellular signaling pathways and their responses to AZD1480 treatment. In contrast with “Typical” B-cell precursor (BCP)-ALL xenograft cells, JAK-mutated xenografts exhibited constitutive JAK pathway activation, as assessed by increased levels of phospho-JAK1 (pJAK1), pJAK2, pSTAT1/3/5, pAKT, pMAP2K1/2 (MEK1/2) and phospho-extracellular signal-regulated kinase 1/2 (pERK1/2). Ex vivo exposure of JAK-mutated xenografts to 1 μM AZD1480 caused rapid (within 1 h) and sustained (up to 24 h) decreases in pSTATs, but minimal reduction in pMEK1/2 and pERK1/2. These results indicate that AZD1480 alone selectively inhibits JAK downstream signaling pathways, which may be insufficient to delay leukemia progression in vivo or induce cell death ex vivo. Moreover, they provide a rationale for dual targeting of the JAK and MAPK pathways to elicit synergistic anti-leukemic cell killing in JAK-mutated ALL. Ex vivo exposure of two JAK2-mutated xenografts to 1 μM of the MEK1/2 inhibitor AZD6244 (selumetinib) caused a profound decrease in pERK1/2, and the combination of 1 μM each of AZD1480 and AZD6244 resulted in reductions of both pSTATs and pERK1/2. Moreover, fixed-ratio MTT cytotoxicity assays using these two JAK2-mutated xenografts demonstrated very strong synergy between AZD1480 and AZD6244, with Combination Indices for each xenograft of 0.36 and 0.098 at the ED50; 0.23 and 0.015 at the ED75; and 0.15 and 0.002 at the ED90. This strong synergistic effect was observed despite AZD1480 and AZD6244 exerting minimal cell killing activity against the xenograft cells when used as single agents. In conclusion, our data indicate that AZD1480 is unlikely to exert significant single-agent activity in the treatment of JAK-mutated pediatric ALL, and that future efforts focusing on dual targeting of the JAK/STAT and MAPK offer a potential pathway to achieving clinical efficacy.