Selective Targeting of JAK/STAT Signaling Is Potentiated by Bcl-Xl Blockade in IL-2–Dependent Adult T-Cell Leukemia

Home , Momelotinib, Pacritinib, Ruxolitinib

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2–dependent adult T-cell leukemia

Meili Zhanga,b, Lesley A. Mathews Grinerc, Wei Jua, Damien Y. Duveauc, Rajarshi Guhac, Michael N. Petrusa, Bernard Wena, Michiyuki Maedad, Paul Shinnc, Marc Ferrerc, Kevin D. Conlona, Richard N. Bamforde, John J. O’Sheaf, Craig J. Thomasc,1, and Thomas A. Waldmanna,1

aLymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bLaboratory Animal Science Program, Leidos Biomedical Research, Inc., Frederick, MD 21702; cDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850; dInstitute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan; eTransponics, Jacobus, PA 17347; and fMolecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892

Contributed by Thomas A. Waldmann, August 21, 2015 (sent for review March 30, 2015)

Adult T-cell leukemia (ATL) develops in individuals infected with 2Rα, IL-15/IL-15Rα) and one paracrine loop that involves IL-9 human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no expression by ATL cells in the early stages of the disease (7–10). curative therapy for ATL. HTLV-1–encoded protein Tax (transacti- These autocrine/paracrine loops are associated with spontaneous vator from the X-gene region) up-regulates Bcl-xL (B-cell lym- ex vivo proliferation of peripheral blood mononuclear cells phoma-extra large) expression and activates interleukin-2 (IL-2), (PBMCs) from ATL patients. Targeted antibodies to IL-2, IL-9, IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified and IL-15 or their receptors significantly inhibit ex vivo pro- JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine- liferation of PBMCs from smoldering/chronic ATL patients (10, dependent ex vivo proliferation of peripheral blood mononuclear 11). These three cytokines share the common γc cytokine re- cells (PBMCs) from ATL patients in smoldering/chronic stages. Cur- ceptor subunit that triggers JAK/STAT signaling, and we noted rently, two JAK inhibitors are approved for human use. In this antiproliferative effects for selected JAK inhibitors in PBMCs study, we examined activity of multiple JAK inhibitors in ATL cell from smoldering/chronic ATL patients (11). On the basis of lines. The selective JAK inhibitor ruxolitinib was examined in a these observations, we have initiated a clinical trial using the high-throughput matrix screen combined with >450 potential JAK inhibitor ruxolitinib for the treatment of ATL. therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was iden- To further explore therapeutic options for ATL, we collected tified as a strong candidate for multicomponent therapy. The com- numerous, matched cell lines representing both cytokine (IL-2)- bination was noted to strongly activate BAX (Bcl-2-associated X dependent reflecting smoldering/chronic ATL and cytokine- independent reflecting acute ATL. We profiled these cell lines protein), effect mitochondrial depolarization, and increase caspase pharmacologically versus a large collection of approved and in- 3/7 activities that lead to cleavage of PARP (poly ADP ribose poly- vestigational drugs. Within this collection were the approved merase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navi- JAK inhibitors ruxolitinib and tofacitinib as well as other nu- toclax independently demonstrated modest antitumor efficacy, merous JAK inhibitors currently being investigated in clinical whereas the combination dramatically lowered tumor burden trials (12). Inhibition of the JAK/STAT pathway has yielded and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients’ PBMCs. These studies provide support for a therapeutic trial in patients Significance with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL. Adult T-cell leukemia (ATL) is caused by the human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative ther- adult T-cell leukemia | Janus kinase | ruxolitinib | navitoclax apy for ATL. In the leukemic cells, the HTLV-1–encoded protein Tax (transactivator from the X-gene region) activates disor- dered interleukin expression, which triggers JAK/STAT sig- -cell leukemia/lymphoma represents ∼10% of lymphoid ma- naling. The HTLV-1–encoded protein Tax also induces the lignancies. Genetic alterations affecting members of the T expression of the antiapoptotic protein Bcl-xL (B-cell lym- Janus kinases (JAKs) and signal transducers and activators of phoma-extra large). In the present study, the combination of transcription (STAT) were discovered in a variety of T-cell the JAK inhibitor ruxolitinib and the Bcl-2/Bcl-xL inhibitor malignancies (1, 2). Furthermore, increases in the common γc navitoclax provided additive/synergistic antitumor efficacy in cytokine concentrations that signal through the JAK1/3, STAT3/5 IL-2–dependent ATL cell lines and in a mouse model of human pathway have been demonstrated in angioimmunoblastic T-cell ATL as well as with ex vivo peripheral blood mononuclear cells lymphoma, gamma delta T-cell lymphoma, and adult T-cell leuke- (PBMCs) from ATL patients compared with either drug alone, mia (ATL), thereby identifying effective therapeutic targets. supporting a therapeutic trial of this combination in patients ATL is an aggressive T-cell malignancy characterized by the + + clonal expansion of CD4 CD25 T lymphocytes that develops in with ATL. a small proportion of individuals infected with human T-cell – Author contributions: M.Z., R.N.B., C.J.T., and T.A.W. designed research; M.Z., L.A.M.G., W.J., lymphotrophic virus-1 (HTLV-1) (3 5). Clinically, ATL is sub- D.Y.D., R.G., M.N.P., B.W., P.S., J.J.O., and C.J.T. performed research; M.M. provided human classified into four subtypes: smoldering, chronic, lymphomatous, ATL cell lines; K.D.C. provided blood samples of ATL patients; M.Z., L.A.M.G., W.J., D.Y.D., and acute (4, 5). Presently, there is no curative therapy for ATL R.G., M.N.P., P.S., M.F., J.J.O., and C.J.T. analyzed data; and M.Z., C.J.T., and T.A.W. wrote (4, 5). In search of effective therapies, we examined key signaling the paper. pathways that confer a proliferation and viability advantage to The authors declare no conflict of interest. – 1 ATL cells. It was noted that the HTLV-1 encoded Tax (trans- To whom correspondence may be addressed. Email: [email protected] or craigt@ activator from the X-gene region) is associated with increased mail.nih.gov. Bcl-xL (B-cell lymphoma-extra large) expression in ATL cells This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (6). Furthermore, we reported two autocrine loops (IL-2/IL- 1073/pnas.1516208112/-/DCSupplemental.

12480–12485 | PNAS | October 6, 2015 | vol. 112 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1516208112 Downloaded by guest on September 27, 2021 therapeutic efficacy in numerous disease indications (11, 13, 14). A B Ruxolitinib Ruxolitinib is approved for myelofibrosis and is being explored 120 – 150 for polycythemia vera (15 17). The therapeutic efficacy of any % Activity drug is also directly related to its specific target portfolio, and 80 (IL2 independent) kinase inhibitors are well established to have diverse selectivities 40 % Activity across the kinome (18). To fully understand the activity of each 100 (IL2 dependent) 0 JAK inhibitor, we performed an in-depth kinome activity profile Lestaurtinib 120 to provide a contextual understanding of selective and non- 50 % Activity Lestaurtinib (IL2 independent) selective antiproliferative actions of each drug. 80 Treatment of cancers is highly reliant on drug combinations, 0 Ruxolitinib 40 % Activity and drug synergies can be manifested through concomitant block- Activity (IL2 dependent) %

% Response(IL2 % Response dependent) ade of parallel signaling pathways or dual inhibition of the same 0 -5-6-7-8-9 pathway at distinct nodes (19). We performed a high-throughput -40 0 40 80 120 log Concentration (M) combination screen to seek out therapeutic agents that combine % Activity (IL2 independent) synergistically with JAK inhibitors to kill human IL-2–dependent ATL cell lines. Several combinations were defined with additive/ CD – + 100 IL-2 independent Ruxolitinib synergistic effects on IL-2 dependent ED40515( ) cells including ED40515(-) C ruxolitinib paired with inhibitors of Bcl-2/Bcl-xL, HDAC, ED41214(-) 80 ST1 mTORC1/2, Aurora A/B/C kinases, PI3Ks, MAPK, and NFkB Su9T01 ATL43Tb(-) pathways. Navitoclax, a Bcl-2/Bcl-xL inhibitor, was identified as MT1 60 ATN1

the most promising candidate for multicomponent therapy. It was IL-2 dependent ED40515(+) Lestaurtinib recently reported that apoptosis induced by JAK2 inhibition was 40 ATL55T(+) ED41214(+) enhanced by the Bcl-2/Bcl-xL inhibitor ABT-737 in mutant JAK2- KOB

% of Control (CPM) LM-Y1 driven malignancies (20, 21). We demonstrated that the combination 20 KKI of ruxolitinib and navitoclax showed additive/synergistic efficacy ATL43T(+) inhibiting proliferation of IL-2–dependent ATL cell lines and 0 0 100 1000 10000 synergy prolonging survival in a murine model of human ATL. IC50 < 2.5 nM Ruxolitinib (nM) Furthermore, the combination regimen synergistically blocked ex IC50 > 2.5 nM vivo proliferation of PBMCs from ATL patients. Our results Fig. 1. Drug response profiles of IL-2–dependent and IL-2–independent provide support for a clinical trial involving the combination – + of JAK and Bcl-xL inhibitors in the treatment of patients with ATL cell lines. (A) Correlation plot of the IL-2 dependent ED40515( )line (x axis) and the IL-2–independent ED40515(–) line (y axis) to the MIPE smoldering/chronic ATL. (Mechanism Interrogation Plate) library of approved and investigational drugs (see ref. 12 for library details). (B) Complete response curves for Results ruxolitinib and lestaurtinib versus the ED40515(+) and ED40515(–) lines. (C) The Antiproliferative Actions of JAK Inhibitors on Human IL-2– Proliferation assay of multiple human IL-2–dependent and IL-2–independent Dependent and IL-2–Independent ATL Cell Lines Are Dependent on ATL cell lines treated with increasing doses of ruxolitinib for 72 h. The assay was Their Polypharmacology. Previously we demonstrated that HTLV- performed in duplicate and repeated. (D) The kinome (polypharmacology) 1–encoded Tax transactivates two autocrine (IL-2/IL-2Rα, profile of ruxolitinib and lestaurtinib (see SI Appendix, Tables S1 and S2 for IL-15/IL-15Rα) loops and one paracrine loop (IL-9) that signal exact values). through the JAK/STAT pathway and that proliferation of ex vivo PBMCs from smoldering/chronic ATL patients were sensitive to – antiproliferative activity versus both IL-2–dependent and IL- JAK inhibition (7 11). To expand the scope of these results, we – B SI Appendix B acquired numerous human ATL cell lines including IL-2–dependent 2 independent cell lines (Fig. 1 and ,Fig.S1 ). and IL-2–independent lines. Western blots showed that all seven IL- When selected agents were examined within two commercially 2–dependent and only one of the six tested IL-2–independent ATL available kinome profiles, the high degree of selectivity reported cell lines express phosphorylated STAT5 (p-STAT5) (SI Ap- for both ruxolitinib and tofacitinib was confirmed while outlining pendix A a more diverse pharmacological profile for lestaurtinib (Fig. 1D , Fig. S1 ). We further examined the response of these SI Appendix cells to a large collection of approved and investigational drugs and , Tables S1 and S2). using a common cell viability assay (12). Single-agent viability was assayed on two IL-2–dependent and three IL-2–independent Matrix Screening Highlights Ruxolitinib Drug Combinations That – ATL cell lines [results are publically available via the Pubchem Block Proliferation and Induce Cell Death in IL-2 Dependent ATL database, assay IDs (AIDs) 1053194 and 1053195; https:// Cells. Using a matrix screening platform, we evaluated rux- × – pubchem.ncbi.nlm.nih.gov/]. The correlation of matched cell olitinib within a 6 6 dose response (matrix) experiment with lines [for instance, ED40515(+) and ED40515(–)] provided an >450 approved or investigational agents (the results are available opportunity to examine specific pharmacologies that di- at https://tripod.nih.gov/matrix-client) (12). Additive/synergistic vergently affect IL-2–dependent and –independent lines (Fig. effects were demonstrated in the IL-2–dependent ED40515(+) 1A). Of particular interest were actions of known JAK in- cell line with ruxolitinib combined with inhibitors of Bcl-2/Bcl-xL hibitors. Compared with IL-2–independent ED40515(–)cells, (navitoclax), HDACs (panobinostat, pracinostat), mTORC1/2 the IL-2–dependent ED40515(+) cells were more sensitive to both (AZD-8055 and torin-2), Aurora A/B/C kinases (AMG-900), PI3Ks ruxolitinib [ED40515(+), IC = 209 nM; ED40515(–), inactive] (Fig. (GSK2126458), and NFκB/IKK (withaferin A, SPC-839, bardox- 50 SI Appendix A 1B) and tofacitinib [ED40515(+), IC50 = 47 nM; ED40515(–), in- olone methyl) ( ,Fig.S2 ). We further evaluated the active] (SI Appendix, Fig. S1B). Further, AZD1480 showed se- combinations of ruxolitinib with AMG-900, GSK2126458, AZD- lective killing of the IL-2–dependent ED40515(+) cells (IC50 = 662 8055, and bardoxolone methyl for their capacity to inhibit cell nM) and not IL-2–independent ED40515(–) cells (SI Appendix, proliferation in five IL-2–dependent and three IL-2–independent Fig. S1B). Thymidine incorporation assays with all 14 ATL cell ATL cell lines (SI Appendix,Fig.S2B). These combination regimens lines showed that IL-2–dependent lines were more sensitive to showed additive/synergistic effects in multiple (if not all) IL- ruxolitinib than IL-2–independent lines (Fig. 1C)andthat 2–dependent ATL lines, whereas none of them showed an AZD1480 showed less selective toxocity to IL-2–dependent lines additive/synergistic effect in the IL-2–independent ATL lines (SI Appendix, Fig. S1C) compared with ruxolitinib (Fig. 1C). tested compared with single drugs alone (SI Appendix,Fig. However, momelotinib, lestaurtinib, and pacritinib all displayed S2B). The combination of ruxolitinib and navitoclax was noted MEDICAL SCIENCES

Zhang et al. PNAS | October 6, 2015 | vol. 112 | no. 40 | 12481 Downloaded by guest on September 27, 2021 to possess additivity/synergy in the IL-2–dependent ED40515(+) A Ruxolitinib (nM) cell line at concentrations that are likely achievable in vivo 11122355461250 as determined by confirmatory 10 × 10 matrix experiments 0011135656625 A 111237101289312 (Fig. 2 ). We further investigated the combination of rux- 2 1 4 3 8 1823242321156 olitinib and navitoclax at multiple doses and their combina- 1 3 6 11 22 34 39 43 47 48 78 tions for 48 h with five IL-2–dependent and three IL-2– 4 8 14 25 36 44 57 66 72 93 39 independent ATL lines for inhibition of proliferation and in- 9 15223140526177939719 duction of cell death (Fig. 2 B–E and SI Appendix,Fig.S2C and D). 11 17 23 35 42 49 62 80 97 100 10 Navitoclax (nM) Five IL-2–dependent ATL lines treated with the combination of 12 17 24 35 43 52 64 89 100 100 5 14 20 26 34 43 52 63 86 95 100 0 ruxolitinib and navitoclax responded in an additive/synergistic 1250 625 312 156 78 39 19 10 5 0 fashion with an enhanced inhibition of cell proliferation as analyzed for 3H-thymidine incorporation (Fig. 2 B–D) and a B ED40515(+) ATL55T(+) dramatic induction of cell death as judged by an increased per- Ruxolitinib (nM) Ruxolitinib (nM) E Navitoclax (nM) Navitoclax (nM) centage of the sub-G1 population (Fig. 2 ). In contrast, this 180000 combination regimen did not possess an additive effect in the IL- 2–independent ATL cell lines tested (SI Appendix,Fig.S2C and 120000 D). Furthermore, these results concurred with the observation that – SI

CPM HTLV-1 encoded Tax increases the transcription of Bcl-xL ( 60000 Appendix, Fig. S1A) (6). Navitoclax inhibited the proliferation of most cell lines tested in this study, regardless of IL-2 dependence 0 0 0 SI Appendix A – 0 0 or independence ( ,Fig.S3 ). The Bcl-2 specific in- 100 50 25 100 25 – 100 10050 hibitor (ABT-199) (22) showed less activity and the Bcl-xL specific 400 200 400 800 400 800 400 200 inhibitor (WEHI 539) (21, 23) showed less or equal activity in inhibition of the proliferation of ATL cell lines compared with C D Control navitoclax (SI Appendix,Fig.S3B). ED41214(+) KOB Ruxolitinib(200nM) Ruxolitinib Navitoclax(2.5nM) Combination 80000 Navitoclax 60000 120 The Combination of Ruxolitinib and Navitoclax Induced Enhanced Combination – 100 Permeabilization of the Mitochondrial Membrane in IL-2 Dependent 60000 45000 80 ATL Cells. Bcl-2 family members include antiapoptotic and

CPM 40000 30000 60 proapoptotic proteins, which have opposing apoptotic activi- 40 ties. Proapoptotic family members Bax and Bak are crucial for in- 20000 15000 duction of permeabilization of the outer mitochondrial membrane % of Control (CPM) 20 (24–26). We evaluated active Bax (Fig. 3A) and mitochondrial 0 0 0 Ruxoli(nM)0 50 200 400 0 100 200 400 800 LM-Y1 membrane potential (Fig. 3B) by flow cytometry. The JC-1 dye Navito(nM)0 25 100 200 0 50 100 200 400 allows for rapid monitoring of mitochondrial polarization through potential-dependent accumulation in mitochondria, Ruxolitinib Navitoclax indicated by a fluorescence emission shift from green (∼529 nm) to E Control (200nM) (100nM) Combination red (∼590 nm) (27). Consequently, mitochondrial depolarization is indicated by a decrease in red/green fluorescence intensity ratio. 4 42638 ED40515(+) Compared with controls, treatment with ruxolitinib (200 nM) for 20 h did not affect active Bax levels (Fig. 3A)andthered/ green JC-1 ratios (Fig. 3B), whereas treatment with navitoclax (100 nM) increased active Bax levels (Fig. 3A) and decreased 5 20 26 65 ATL55T(+) red/green JC-1 ratios (Fig. 3B). Furthermore, cells treated with the combination of ruxolitinib and navitoclax or cells cultured without IL-2 in combination with navitoclax showed significantly higher levels of active Bax and lower red/green JC-1 ratios 6 32950ED41214(+) compared with cells treated with either drug alone (Fig. 3 A and B).

The Combination of Ruxolitinib and Navitoclax Induced Caspase 3/7 Activation, Which Resulted in Cleavage of PARP and Mcl-1 in IL-2– 4 10 27 56 KOB Dependent ATL Cells. Once activated, Bax and Bak induce permeabilization of the mitochondrial outer membrane and sub- sequent release of apoptogenic molecules such as cytochrome c, which leads to caspase activation (24, 26). Activated caspases, in 7274688LM-Y1 turn, cleave substrates including PARP and Mcl-1. Ruxolitinib Counts (Navito, treatment (200 nM) showed slightly increased caspase 3/7 activity 10nM) and PARP cleavage, whereas treatment with navitoclax (100 nM) DNA content significantly increased caspase 3/7 activity and PARP cleavage in the ED40515(+) and ATL55T(+) cell lines (Fig. 3 C and D and SI Fig. 2. The antiproliferative activity of ruxolitinib and navitoclax in combi- Appendix, Fig. S4). A combination of ruxolitinib and navitoclax nation is additive/synergistic in IL-2–dependent human ATL cell lines. (A)A10× significantly increased caspase 3/7 activity and PARP cleavage in 10 matrix block experiment determining the viability of the IL-2–dependent ED40515(+) and ATL55T(+) cell lines compared with either ED40515(+) line as judged by CellTiter-Glo. (B) Proliferation assay of the IL-2– C D SI Appendix + + drug alone (Fig. 3 and and ,Fig.S4). Additionally, dependent ED40515( )andATL55T( ) lines treated with ruxolitinib (pink bars), the combination regimen was associated with significant cleavage navitoclax (blue bars), or in combination (green bars) in a multiple dose– response plot for 48 h as judged by [H3]Thymidine incorporation. The data were a representative of three different experiments. (C) Proliferation assay of the IL-2–dependent ED41214(+) and KOB lines treated with ruxolitinib, navi- navitoclax, or in combination at a single dose for 48 h. The data in C and D toclax, or in combination in a selective dose–response plot for 48 h. (D)Pro- were expressed as mean± SD (n = 3). (E)DNAcontentofhumanIL-2–de- liferation assay of the IL-2–dependent LM-Y1 line treated with ruxolitinib, pendent ATL cell lines treated as indicated for 48 h.

12482 | www.pnas.org/cgi/doi/10.1073/pnas.1516208112 Zhang et al. Downloaded by guest on September 27, 2021 We did not demonstrate any decreased expression of antiapoptotic A Control Ruxolinib Navitoclax Combination WO IL-2 WO IL-2+Navito D01()AL5()ED41214(+) ATL55T(+) ED40515(+) proteins after ruxolitinib treatment or following IL-2 withdrawal (SI Appendix B 2 317292 37 , Fig. S5 ). The general similarity in protein expression changes between ruxolitinib treatment and IL-2 withdrawal suggests that cellular responses were associated with blockade of JAK/STAT signaling rather than off-target drug effects. These 39 2 310313 results suggested that different cell lines showed expression changes of different proapoptotic members following ruxolitinib treatment, which might be associated with the changes in relative abundance and functional activity of Bcl-2 family members and 2 2 10 19 2 22 thereby prime cells for an enhanced response to navitoclax. Counts The Combination of Ruxolitinib and Navitoclax Significantly Inhibited Bax Tumor Growth and Resulted in Prolonged Survival of Tumor-Bearing B C Mice. Human IL-2–dependent ATL cell lines represent a model Control Ruxolitinib Navitoclax Combination of smoldering/chronic forms of ATL, which are clinical disease

ED40515(+) Control Navitoclax Ruxolitinib Combination stages that manifest constitutive JAK/STAT activation and spon- 16 taneous ex vivo proliferation of ATL cells. These cell lines require a 5 74058 supply of extrinsic human IL-2 for their survival in vitro. To es- tablish a murine model of human IL-2–dependent ATL to evaluate T5T+ ED41214(+) ATL55T(+) 12 potential therapies, we transfected ED40515(+) cells with the

4 71668 construct pMXs-IRES-huIL2-GFP and established a cell line 8 termed ED(+)/IL-2 that is capable of in vitro viability without extrinsic IL-2. The ED(+)/IL-2 cells express high levels of the 4 IL-2 gene as detected by real-time PCR (SI Appendix, Fig. S6A),

Fold changes over control although the IL-2 protein was not detectable in the culture su- 12822 JC-1 red 0 pernatant. Flow cytometric analysis showed that the ED ED40515(+)ATL55T(+) (+)/IL-2 cells expressed intracellular IL-2 similarly to the original JC-1 green ED40515(+) cells when cultured with IL-2 (SI Appendix,Fig.S6B). Both the ED(+)/IL-2 and the original ED40515(+) cell lines D ED40515(+) ATL55T(+) responded to ruxolitinib treatment similarly, as demonstrated by 24h 48h 24h 48h inhibition of proliferation (SI Appendix, Fig. S6C) and reduced Parp expression of p-STAT5 and p-STAT3 (SI Appendix, Fig. S6D). Cleaved Parp Therapy in this ED(+)/IL-2 model was initiated (n = 8–9) when tumors were established (average tumor volume of Mcl-1 100 mm3). Treatment of ED(+)/IL-2 tumor-bearing NOD/SCID/ − − γ / (NSG) mice with either ruxolitinib at a dose of 50 mg/kg/ d by continuous infusion pump for 14 d or navitoclax at a dose of Cleaved Mcl-1 40 mg/kg/d orally for 6 d significantly delayed tumor growth (Fig. 4A and SI Appendix, Fig. S6E) and prolonged survival of Actin tumor-bearing mice (Fig. 4B, P < 0.001) compared with control. 1 234 1234 1234 12 34 Combination therapy with ruxolitinib and navitoclax provided greater therapeutic efficacy as judged by tumor volumes (Fig. 4A 1. Control; 2. Ruxolitinib (200nM); SI Appendix E B 3. Navitoclax (100nM); 4. Ruxolitinib+navitoclax and ,Fig.S6 ) and prolongation of survival (Fig. 4 , P < 0.001) compared with monotherapies. All of the mice in the Fig. 3. The combination of ruxolitinib and navitoclax triggers the mito- control, ruxolitinib, and navitoclax monotherapy groups died from chondrial apoptosis pathway in human IL-2–dependent ATL cells. (A) Flow tumor progression by day 30. In contrast, 75% of the mice in the cytometry analysis of intracellular active Bax at 20 h after different treatments as indicated. (B) Flow cytometry analysis of mitochondrial membrane potential assessed by JC-1 staining at 24 h after different treatments as indicated. (C) Caspase 3/7 activities measured by Caspase-Glo assay kit at 24 h AB after different treatments as indicated. This assay was performed in tripli- 1800 100 cate, and the data were expressed as mean± SD. (D) Western blot analysis of 1500 cleavage of PARP and Mcl-1 proteins at 24 or 48 h after different treatments ) 3 80 as indicated. The concentrations of ruxolitinib and navitoclax used in the 1200 studies shown in this figure were 200 nM and 100 nM, respectively. 60 Control 900 Ruxolitinib 40 Navitoclax 600 of Mcl-1 from a 40-kDa antiapoptotic to a 24-kDa proapoptotic Combination Tumor volume (mm Tumor form (28), especially in ATL55T(+) cells (Fig. 3D). 300 % of mice surviving 20 Several lines of evidence indicate that activation of the JAK/ 0 0 STAT pathway up-regulates the expression of antiapoptotic 005101520 25 10 20 30 40 50 Days after initiation of therapy Days after initiation of therapy Bcl-2 family proteins and that inhibition of this pathway induces expression of proapoptotic BH3-only proteins, thereby inducing Fig. 4. The combination therapy of ruxolitinib and navitoclax significantly apoptosis (13, 20, 21). Treatment with ruxolitinib or IL-2 with- inhibits ED(+)/IL-2 tumor growth in vivo. Female NSG mice were injected s.c. 7 drawal reduced expression of p-STAT5 in all IL-2–dependent with 1 × 10 ED(+)/IL-2 cells. When the average tumor volume reached about 3 = – ATL cell lines (SI Appendix, Fig. S5A). Up-regulation of active 100 mm , the tumor-bearing mice were divided into four groups (n 8 9) with comparable average tumor volumes, and therapy was started. Rux- Bim (BimEL) was observed in ED40515(+) and ED41214(+), + + olitinib was continuously administered by an s.c. infusion pump at a dose of and Puma was up-regulated in ED40515( ), ED41214( ), and 50 mg/kg/d for 14 d, and navitoclax was given orally at a dose of 40 mg/kg/d for KOB cell lines (SI Appendix, Fig. S5B). Phosphorylation of Bad 6d.(A) Average tumor volumes during the therapeutic course for each group. was reduced following the same treatments (SI Appendix,Fig.S5B). (B)Kaplan–Meier survival plot of the mice in the therapeutic study. MEDICAL SCIENCES

Zhang et al. PNAS | October 6, 2015 | vol. 112 | no. 40 | 12483 Downloaded by guest on September 27, 2021 combination group were alive at that time (Fig. 4B). We repeated for ATL. On the basis of previous studies and the work pre- the therapeutic study (n = 2–4) for a short period using the same sented here, we propose that JAK inhibition should be included tumor model, doses, and dosing schedule, with the exception that as a component of a therapeutic regimen for smoldering/chronic ruxolitinib was administered for 6 d and the experiment was ter- ATL. Previously, we demonstrated that HTLV-1 Tax trans- minated on day 6 after therapy (SI Appendix,Fig.S6F–I). Mono- activates two autocrine loops (IL-2/IL-2R, IL-15/IL-15R) and therapy with ruxolitinib or navitoclax significantly inhibited tumor one paracrine loop (IL-9) of γc cytokines that signal through growth, as demonstrated by a reduction in tumor volume and JAK1/3 (7–10). Furthermore, ex vivo spontaneous proliferation weight compared with controls (SI Appendix,Fig.S6F–H). Com- of PBMCs from smoldering/chronic ATL patients was blocked bination treatment yielded a significantly greater therapeutic efficacy by the JAK inhibitor tofacitinib (11). In this study, we profiled an (SI Appendix,Fig.S6F–H) compared with monotherapies. Analysis expanded set of ATL cell lines including matched lines repre- of platelet levels (an established side effect of navitoclax) demon- senting cytokine-dependent and -independent models of ATL strated a significant reduction in circulating platelets associated with versus a large library of drugs. These studies confirmed the ef- navitoclax treatment (SI Appendix,Fig.S6I). fectiveness of ruxolitinib and tofacitinib for IL-2–dependent cell lines (Fig. 1B and SI Appendix, Fig. S1B). A broader profile of The Combination of Ruxolitinib and Navitoclax Significantly Inhibited several investigational JAK inhibitors highlighted the pro- 6-d ex Vivo Spontaneous Proliferation of PBMCs from Patients with miscuous nature of momelotinib, lestaurtinib, pacritinib, and Smoldering/Chronic ATL. Analyses of drug responses using in vitro fedratinib that is likely responsible for their activity in non-JAK/ – B SI Appendix B culture and murine models represent good but often compromised STAT driven cancer lines (Fig. 1 and , Fig. S1 ). Activity of ruxolitinib in IL-2–dependent ATL cell lines (Fig. facsimiles of human disease states. Spontaneous proliferation of ex C vivo PBMCs from patients with smoldering/chronic ATL represents 1 ), in the mouse model of human ATL (Fig. 4), and with ex an attractive model to assess the therapeutic potential of drug vivo PBMCs from a new group of smoldering/chronic ATL pa- combinations. Proliferations of ATL cells in 6-d cultures assessed by tients (Fig. 5) further highlighted our interest in this agent. 3H-thymidine uptake were determined for PBMCs from five Given the potential of JAK inhibitors in preclinical ATL studies, patients with smoldering/chronic ATL who were in the autocrine we reasoned that JAK inhibitor-based multicomponent therapies IL-2–dependent phase of their disease. Spontaneous proliferation may yield clinical efficacy. We advanced ruxolitinib to a matrix combination study with >450 additional drugs. This analysis of PBMCs was partially inhibited by addition of individual an- + tibodies specifically directed toward IL-2 and, to a lesser extent, highlighted several ruxolitinib drug combinations that dem- toward IL-9, IL-15 (SI Appendix, Fig. S7). Profound inhibition of onstrated synergy/additivity. We advanced the combination of ex vivo proliferation was achieved by simultaneous addition of ruxolitinib and the Bcl-2/Bcl-xL inhibitor navitoclax for additional studies. A confirmatory 10 × 10 matrix analysis in the IL- antibodies directed against all three cytokines (SI Appendix, Fig. 2–dependent ED40515(+) cell line highlighted the synergistic/ S7). Addition of ruxolitinib or navitoclax inhibited 6-d ex vivo additive nature of this combination (Fig. 2A). Furthermore, this proliferation of PBMCs from these patients (Fig. 5). Moreover, a combination maintained efficacy in multiple IL-2–dependent ATL combination of ruxolitinib and navitoclax provided enhanced cell lines tested (Fig. 2 B–D). It is critical to note that the Bcl-2– inhibition of ex vivo proliferation of PBMCs from the five pa- specific inhibitor ABT-199 showed much less efficacy with ATL cell tients by 90%, a significantly greater inhibition compared with SI Appendix B B P < lines compared with navitoclax ( ,Fig.S3 ). This ob- either drug alone (Fig. 5 , 0.01). servation is in accord with the demonstration that HTLV-1–enco- Discussion ded Tax in ATL activates the expression of Bcl-xL but not Bcl-2 (6). To better understand the mechanistic basis of additive/syner- Treatment options for ATL patients are extremely limited. gistic effects observed, we examined several key elements of each Standard combination chemotherapy regimens (e.g., CHOP) drug’s primary effect on ATL cells. Ruxolitinib treatment ef- that remain effective in B-cell lymphomas are relatively in- fectively blocked STAT5 phosphorylation in IL-2–dependent effective. A meta-analysis suggested that AZT therapy in com- lines and additionally lead to expression changes of different bination with IFN may possess value, although morbidity and a proapoptotic members, including Bim, Puma, and p-Bad, in lack of efficacy in patients with prior chemotherapy remain (29, different IL-2–dependent cell lines (SI Appendix, Fig. S5). These 30). Antibody-based approaches including use of daclizumab, outcomes might prime IL-2–dependent lines to respond to the alemtuzumab, and anti-CCR4 showed value but are not curative Bcl-2/Bcl-xL inhibition by navitoclax. Furthermore, the combi- (31–34). Allogeneic bone marrow transplantation is an aggres- nation strikingly increased the levels of active Bax and enhanced sive approach that is curative in select patients (35). Due to these permeabilization of mitochondrial membranes. Consequently, limited options, there is a clear need for new treatment strategies caspase 3/7 activation and an associated increase in PARP and Mcl-1 cleavage occurred upon cotreatment with ruxolitinib and navatoclax. Aspects of these results have been noted in other disorders (13, 20, 21, 36). Gozgit et al. reported that inhibition of ABRuxolitinib (nM) Navitoclax (nM) P < 0.01 JAK2 blocked production of the Pim1/2 kinases and resulted in a 100 corresponding decrease in BAD phosphorylation (13), and Will 10000 P = 0.46 8000 et al. demonstrated that apoptosis induced by JAK2 inhibition is 80 6000 mediated by Bim and enhanced by the BH3 mimetic ABT-737 in JAK2 mutant human erythroid cells (20). Chen et al. demon-

CPM 4000 60 strated that triptolide inhibited JAK2 transcription and induced 2000

% of inhibition apoptosis in human myeloid proliferative disorder cells bearing 0 40 0 0 JAK2 V617F through caspase-mediated cleavage of Mcl-1 (36). 50 50 100 100 20 Recently, it was reported that combined targeting of JAK2 and 200 200 Ruxolitinib Navitoclax Combination Bcl-2/Bcl-xL mediated prolonged disease regressions and cures Fig. 5. Therapeutic effects of ruxolitinib and navitoclax on the 6-d ex vivo in mice bearing JAK2 mutant tumors (21). Our studies and these spontaneous proliferation of PBMCs from patients with smoldering/chronic reports suggest that disruption of JAK signaling in several he- ATL. (A) Antiproliferative effects on PBMCs from an ATL patient cultured ex matological malignancies leads to a generalized increase in proa- vivo for 6 d in the presence of increasing concentrations of ruxolitinib (pink), poptotic signatures that can enhance actions of antiapoptotic navitoclax (blue), or their combinations (green). (B) The percent inhibition of member inhibitors like navatoclax. The clinical potential of proliferation of PBMCs from the five patients by ruxolitinib (200 nM), navi- navitoclax is limited due to thrombocytopenia associated with toclax (100 nM), or their combinations. The experiments were performed in Bcl-xL inhibition of platelets. A selective Bcl-2 inhibitor (ATB- triplicate, and the data were expressed as means. 199) was demonstrated to maintain an antiproliferative activity

12484 | www.pnas.org/cgi/doi/10.1073/pnas.1516208112 Zhang et al. Downloaded by guest on September 27, 2021 in selected cancers without thrombocytopenia (22). Our exami- Materials and Methods nation of this agent demonstrated a profoundly inferior activity More materials and methods are described in SI Appendix, SI Materials profile (SI Appendix, Fig. S3B), suggesting inhibition of Bcl-xL is and Methods. critical for efficacy in ATL. A key aspect of all preclinical studies remains in demonstra- High-Throughput Screening Platform for Identification of Drug–Drug tion of in vivo efficacy. Human IL-2–dependent ATL cells will Combinations for Human IL-2–Dependent ATL Therapy. The single agent and not grow in mice. We therefore established a mouse model of combination high-throughput assessments of the matched IL-2–dependent human IL-2–dependent ATL via a transfected ED40515(+)cellline and IL-2–independent ATL cell lines were performed as described (12). with a construct encoding human IL-2. Evaluation of monotherapy with ruxolitinib or navitoclax in this model demonstrated delayed Mouse Model of ED(+)/IL-2 and Therapeutic Study. An ED(+)/IL-2 cell line was tumor growth and prolonged survival of tumor-bearing mice established as described in SI Appendix, SI Materials and Methods. The xe- compared with controls. Critically, combination therapy yielded nograft tumor model of human IL-2–dependent ATL was made by s.c. in- 7 far greater therapeutic efficacy than monotherapies in terms of jection of 1 × 10 ED(+)/IL-2 cells into the right flank of female NSG mice both tumor volume and prolongation of survival (Fig. 4). (Jackson Labs). The therapeutic protocol is described in SI Appendix, SI Ma- Beyond demonstration of efficacy within in vitro and in vivo terials and Methods. All animal experiments were approved by the National (nonhuman) models, it remained important to validate key re- Cancer Institute (NCI) Animal Care and Use Committee and were performed in accordance with NCI Animal Care and Use Committee guidelines. sults in primary human disease samples. To obtain insight into the value of the ruxolitinib/navitoclax combination, we examined Ex Vivo Cultures of PBMCs from ATL Patients. Smoldering/chronic ATL patient spontaneous proliferation of ex vivo cultures of PBMCs from blood samples were obtained from patients under the care of the Clinical Trials smoldering/chronic ATL patients. Addition of ruxolitinib or navi- Team, Lymphoid Malignancies Branch, NCI. This study protocol was approved toclax inhibited 6-d ex vivo spontaneous proliferation of PBMCs by the Institutional Review Board of the NCI. Informed consent was obtained from the five patients (Fig. 5). Furthermore, the combination in writing in accordance with the Declaration of Helsinki. The proliferation provided greater inhibition of the 6-d proliferation of the assay of ex vivo 6-d culture was performed as described previously (11). PBMCs (P < 0.01) compared with either drug alone (Fig. 5). The present study demonstrated that the combination of ACKNOWLEDGMENTS. This research was supported by the Division of ruxolitinib and navitoclax provided additive/synergistic activity in Preclinical Innovation, National Center for Advancing Translational Sciences; IL-2–dependent ATL cell lines and in a mouse model of human the Molecular Libraries Initiative of the National Institutes of Health IL-2–dependent ATL as well as on ex vivo 6-d cultures of Roadmap for Medical Research; the Intramural Research Programs of the National Center for Advancing Translational Sciences, National Human PBMCs from ATL patients. These findings provide support for a Genome Research Institute and National Cancer Institute (NCI), Center for therapeutic trial in patients with smoldering/chronic ATL using a Cancer Research. This project has been funded in part with federal funds combination regimen that inhibits JAK1 and the Bcl-xL. from the NCI, NIH, under Contract N01-CO-12400.

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