Combined Targeting of JAK2 with a Type II JAK2 Inhibitor and mTOR with a TOR Kinase Inhibitor Constitutes Synthetic Activity in JAK2-Driven Ph-like Acute Lymphoblastic Leukemia

Background and rationale: Philadelphia chromosome-like acute lymphoblastic leukemia (“Ph-like ALL”) is a subtype of high-risk B-precursor ALL (B-ALL), which carries a high risk of relapse with conventional chemotherapy(Roberts et al, N Engl J Med. 2014). Rearrangements in CRLF2, leading to overexpression of cytokine receptor for thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALL and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010;Tasian et al, Blood 2014).In addition,JAK2 fusion proteins, such as PAX5-JAK2 represent a novel class of JAK2-driven cellular transformation in B-ALL(Dagmar et al, Blood 2015). Our prior studies in Ph+ B-ALL established that combining tyrosine kinase inhibitors (TKIs) with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs)provides greater anti-leukemia efficacy compared to TKIs in Ph+ ALL (Janeset al, Nat. Med. 2013). In this study, we investigated anti-leukemia efficacy and intracellular signaling networks upon combination of type I or type II JAK2 inhibitors and TOR-KIs in JAK2-driven Ph-like ALL models. 

Methods. The human B-precursor Ph-like ALL cell lines MUTZ5 (which harborsIGH-CRLF2translocation and JAK2 R683G mutation), MHH-CALL-4 (IGH-CRLF2 translocation and JAK2 I682F),Reh (ETV6-RUNX1 B-precursor ALL cell line)and mouse Arf-null PAX5-JAK2-MIG + IK6-MIR(IL7-dependent primary Arf^-/- pre-B cells expressing the dominant negative Ikaros isoform IK6 with PAX5-JAK2 fusion protein) were studied. Signal transduction inhibitors (STIs): JAK2 type I inhibitor ruxolitinib and type II inhibitor NVP-BBT594 (Andraos et al., Cancer Discovery 2012); allosteric mTOR inhibitor rapamycin or mTOR-KI AZD2014. Effects on intracellular signaling were determined using phospho-flow cytometry and Westernblot analysis. Anti-leukemia effects were quantified using CellTiter-Glo viability assay and annexin V flow cytometry.

Results.  In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling(Fig 1D). JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, ERK and S6 activation, yet failed to affect4E-BP1 activation. The TOR-KI AZD2014 but not rapamycin fully inhibited phosphorylation of 4E-BP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth inhibition of Ph-like cells. Combination of ruxolitinib and AZD2014 further inhibited cell proliferation, yet did not induce apoptotic cell death. Recent studies indicate persistence of JAK2-mutated cells upon chronic exposure to type I JAK2 inhibitors, through an adaptive resistance mechanism involving 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 p-STAT5; in contrast, BBT594 diminished bothp-JAK2 and p-STAT5 in both cell lines. Unexpectedly, BBT594 induced apoptotic cell death in all JAK2-driven Ph-like ALL cell linesMUTZ5, MHH-CALL-4 and Arf-null PAX5-JAK2+IK6, but not in REH cells. Combination of BBT594 with AZD2014 further inhibited phosphorylation of JAK2, AKT, 4E-BP1 and eIF4E, and synergistically induced apoptosis and reduced cell viability in Ph-like ALL cell lines(combination index: MUTZ5, 0.71; MHH-CALL-4, 0.57; Arf-nullPAX5-JAK2+ IK6, 0.81). Of importance, BBT594 and AZD2014 combination induced apoptosis in five JAK2-mutant Ph-like ALL xenograft  primary samples.

In summary, these results suggest that efficient blockade of JAK2/STAT5 with type II JAK2 inhibitors translates into cell death of mutant JAK2-driven Ph-like ALL cells. Furthermore, concomitant blockade of TORC1 signaling with TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4E-BP1 and causes synthetic activity, providing avenues for novel rationally designed combinatorial regimens in this subset of Ph-like B-ALL. The in vivo studies to test these hypotheses are ongoing using patient-derived xenografts.