Dual Targeting of JAK2 Signaling with a Type II JAK2 Inhibitor and of mTOR with a TOR Kinase Inhibitor Induces Apoptosis in CRLF2-Rearranged Ph-like Acute Lymphoblastic Leukemia

Abstract

Philadelphia chromosome-like acute lymphoblastic leukemia (“Ph-like ALL”) is a subtype of high-risk B-precursor ALL (B-ALL) that carries a high risk of relapse after conventional chemotherapy (Mullighan et al, N Engl J Med. 2009). Rearrangements in CRLF2, leading to overexpression of the receptor for the cytokine thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALLs and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010; Tasian et al, Blood 2014). Previous studies established that combining a tyrosine kinase inhibitor (TKI) with an mTOR inhibitor provides greater anti-leukemia efficacy than a TKI alone in Ph+ B-ALL (Janes et al, Nat. Med. 2013). While allosteric mTOR inhibitors such as rapamycin only partially block mTORC1 and do not directly inhibit mTORC2, second-generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) efficiently block both mTOR outputs and show greater efficacy when combined with TKIs. In this study, we investigated anti-leukemia efficacy and intracellular signaling networks in Ph-like CRLF2+ ALL models treated with combinations of a type I or type II JAK-2 inhibitor and a TOR-KI.The inhibitors were tested in human B-precursor Ph-like ALL cell lines MUTZ5 (IGH@-CRLF2 translocation, JAK2 R683G mutation) and MHH-CALL-4 (IGH@-CRLF2 translocation, JAK2 I682F mutation), B-ALL cell line REH (CRLF2wt), and primary CRLF2+ xenograft cells in vitro. For signaling and growth inhibition studies, cells were stimulated with 25 ng IL-7 or TSLP for 30 min, then with JAK2 type I inhibitor ruxolitinib (500nM) or type II inhibitor NVP-BBT594 (500nM) (Andraos et al., Cancer Discov. 2012) and allosteric mTOR inhibitor rapamycin or TOR-KI AZD2014. Effects on intracellular signaling were determined by phospho-flow cytometry. Anti-leukemia effects were characterized by viable cell counts and annexin V flow cytometry.In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (p-JAK2(Tyr1008), p-STAT5(Ty694)) and AKT/pS6 signaling (p-AKT(Ser473), p-rS6(S235/236) (Fig. 1A). Stimulation with IL-7, mimicking support by the normal bone marrow environment, induced a lesser degree of activation of these phospho-proteins, except for p-4EBP1(T37/46), which was constitutively highly expressed in these cells and further induced by IL-7. These findings warranted combination studies of JAK2 and mTOR inhibitors. JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, and S6 activation, yet failed to decrease p-4EBP1 (Fig. 1A). AZD2014 but not rapamycin fully inhibited p-4EBP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth arrest in CRLF2+ cells (Fig. 1A, C). In turn, combination of ruxolitinib and AZD2014 further reduced cell proliferation but did not induce apoptotic cell death (Fig. 1B, D).Recent studies indicate persistence of JAK2-mutated cells in myeloproliferative neoplasms upon long-term exposure to a type I JAK2 inhibitor, mediated by JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2, in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of STAT5 phosphorylation; in contrast, BBT594 diminished both p-JAK2 and p-STAT5. Unexpectedly, BBT594 induced apoptotic cell death in both MUTZ5, MHH-CALL-4 (Fig 1B) and in ALL blasts recovered from primary CRLF2+ xenograft and grown in OP9 in vitro co-culture; the combination of BBT594 with AZD2014 increased apoptosis and reduced cell viability even further, in both cell lines and in stroma-attached primary ALL cells.In summary, these results suggest that efficient blockade of JAK2/STAT5 with a type II JAK2 inhibitor translates into cell death of JAK2-addicted CRLF2-rearranged cells and may have the capacity to eliminate JAK2-mutated clones. Concomitant blockade of TORC1 signaling with a TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4EBP1 and causes synthetic lethality, providing avenues for novel, rationally designed combinatorial regimens in this subset of Ph-like B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.