may have altered the results of coagulationparameters, which did not show significantassociations with early HD. Furthermore,the analysis of data from clinical trials, whichinvolve patient selection, may not begeneralizable to the entire APL population.Despite these limitations, this is the largest dataset of patients and clinical outcomes in APL thatis available at this time. The clear-cut primaryend point of early HD in this study guaranteesthe standardization and quality of data.

In conclusion, there is a need to identifyreliable risk factors of early HD in APL. Thestudy byMantha and colleagues identifies highWBC count as an independent predictor ofHD, and supports the inclusion of performancestatus in future risk profiles.

Future studies should identifywhether circulating plasma biomarkers of

hypercoagulation and/or hyperfibrinolysis(see figure) or global rapid coagulation

assays (ie, thrombin generation, orthromboelastography) can add value to thepredictive model of early HD. Finally, theevaluation of efficacy and safety of new oralanticoagulants and LMWHs in reducing HDrisk should be considered.Conflict-of-interest disclosure: The author

declares no competing financial interests. n

REFERENCES1. Mantha S, Goldman DA, Devlin SM, et al.Determinants of fatal bleeding during induction therapyfor acute promyelocytic leukemia in the ATRA era. Blood.2017;129(13):1763-1767.

2. Rickles FR, Falanga A, Montesinos P, Sanz MA,Brenner B, Barbui T. Bleeding and thrombosis in acuteleukemia: what does the future of therapy look like?Thromb Res. 2007;120(2 Suppl 2):S99-S106.

3. Breccia M, Lo Coco F. Thrombo-hemorrhagic deathsin acute promyelocytic leukemia. Thromb Res. 2014;133(2 Suppl 2):S112-S116.

4. Kwaan HC, Cull EH. The coagulopathy in acutepromyelocytic leukaemia–what have we learned in the pasttwenty years. Best Pract Res Clin Haematol. 2014;27(1):11-18.

5. Tallman MS, Brenner B, Serna JL, et al. Meetingreport. Acute promyelocytic leukemia-associatedcoagulopathy, 21 January 2004, London, United Kingdom.Leuk Res. 2005;29(3):347-351.

6. Falanga A, Iacoviello L, Evangelista V, et al. Loss ofblast cell procoagulant activity and improvement ofhemostatic variables in patients with acute promyelocyticleukemia administered all-trans-retinoic acid. Blood. 1995;86(3):1072-1081.

7. Altman JK, Rademaker A, Cull E, et al. Administrationof ATRA to newly diagnosed patients with acutepromyelocytic leukemia is delayed contributing to earlyhemorrhagic death. Leuk Res. 2013;37(9):1004-1009.

8. Sanz MA, Grimwade D, Tallman MS, et al.Management of acute promyelocytic leukemia:recommendations from an expert panel on behalf of theEuropean LeukemiaNet. Blood. 2009;113(9):1875-1891.

DOI 10.1182/blood-2017-02-763490

© 2017 by The American Society of Hematology

l l l LYMPHOID NEOPLASIA

Comment on Gayle et al, page 1768

Toward autophagy-targetedtherapy in lymphoma-----------------------------------------------------------------------------------------------------

Lapo Alinari THE OHIO STATE UNIVERSITY

In this issue of Blood, Gayle et al provide evidence that apilimod, a potent andselective phosphatidylinositol-3-phosphate 5 kinase (PIKfyve) inhibitor, inducessignificant cytotoxicity at clinically achievable concentrations in preclinical modelsof B-cell non-Hodgkin lymphoma (NHL) via inhibition of the autophagy flux andperturbation of lysosome homeostasis.1

Induction of prothrombotic vascular endothelium. The APL cell and hemostasis: APL cells express procoagulant factors

(ie, tissue factor, cancer procoagulant, procoagulant microparticles), fibrinolytic proteins (ie, plasminogen activators

[u-PA, t-PA] and inhibitors [PAI-1] and their receptors [u-PA, annexin II]), and nonspecific proteases (ie, elastase),

which activate coagulation and fibrinolysis. In addition, these cells possess an increased capacity to adhere to the

vascular endothelium, and secrete inflammatory cytokines (ie, interleukin-1b [IL-1b] and tumor necrosis factor a

[TNF-a]), which downstream stimulate the expression of prothrombotic properties of endothelial cells, leukocytes, and

platelets. All of these events are reflected in the peripheral blood by alterations in the levels of circulating biomarkers of

hypercoagulation, hyperfibrinolysis, proteolysis, and inflammation. u-PAR, urokinase-type plasminogen activator

receptor; VEGF, vascular endothelial growth factor. Professional illustration by Somersault18:24.

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Autophagy is a highly conserved sequentialcatabolic process that occurs at basal levelsin healthy cells and allows them to sequesterand degrade faulty proteins and damagedconstituents in autophagolysosomes.2

Degradation ultimately occurs by exposingthe cargo to the catalytic activity of lysosomalproteases (cathepsins). Accumulating evidencesupports the role of enhanced autophagyduring the neoplastic transformation process,as well as in the progression of alreadyestablished neoplasms, by promoting cellsurvival under adverse conditions such ashypoxia and nutrient deprivation throughthe recycling of metabolic precursors andelimination of cellular debris.3 In support ofthe role of autophagy in cancer, genetic andpharmacologic inhibition of this processin different tumor types, including NHL,promotes tumor cell death, suggesting that theadministration of autophagy inhibitors may

be beneficial to cancer patients.4,5 Chloroquineand hydroxychloroquine are 2 autophagy fluxinhibitors tested in early phase clinical trials insolid tumors and hematologic malignancies;however, low potency and off-target effectshave limited their clinical development,highlighting the need for more selective andpotent inhibitors of autophagy.6

PIKfyve is an endosomal lipid kinasethat phosphorylates P1(3)P to yieldphosphatidylinositol 3,5-bisphosphate (PI[3,5]P2).PIKfyve-mediated PI(3,5)P2 signaling hasbeen shown to play a critical role in multiplebiological processes, including autophagy, byregulating endosomal membrane trafficking.7

Apilimod mesylate is an orally bioavailablesmall molecule initially developed as aninterleukin-12 (IL-12) and IL-23 inhibitorand evaluated in clinical trials in patients withinflammatory diseases.8 In this issue of Blood,Gayle et al identified apilimod among the

most potent drugs in a library of clinicallyrelevant compounds using a high-throughputscreening assay. After the initial screen, theantiproliferative activity of apilimod was testedin cell lines derived from different tumor types.B-cell NHL cells were identified as the mostbroadly sensitive. Interestingly, apilimod at lownanomolar concentrations induced significantcell death in mantle cell lymphoma, germinalcenter, activated B-cell, and myc-drivendiffuse large B-cell lymphomas (DLBCL).Cytotoxic activity of apilimod appeared to beselective against malignant B cells, as a varietyof normal cells including B cells and tissuesfrom healthy donors were highly resistant toapilimod. Furthermore, apilimod showedsignificant antitumor activity in xenograft aswell as syngeneic mouse lymphoma models.Using capture mass spectrometry and a geneticapproach, the authors showed PIKfyve tobe the critical target for apilimod-mediatedB-cell NHL cell death. Their data show thattreatment of lymphoma cell lines with apilimodinduces p62 and LC3-II accumulation thatis further increased by cotreatment withrapamycin, an autophagy inducer, thussuggesting blockage of the autophagy flux.Treatment with apilimod was associated withenlargement of the lysosomal compartment andincrease of pro- (inactive) cathepsin levelswithout lysosomal membrane permeabilizationand mature (active) cathepsin accumulationin the cytosol, indicating a noncanonicalmechanism of cell death at the lysosomal level.Interestingly, the authors showed that, asa consequence of PIKfyve inhibition,transcription factor EB (TFEB), whichis highly expressed in B-cell NHL cells,was translocated to the nucleus in itsunphosphorylated/active form where itpromoted the transcription of its target geneCLCN7. A genome-wide CRISPR screenidentified OSTM1 and SNX10 as keygenes involved in apilimod-mediated celldeath. Both CLCN7 and OSTM1 encode aCl2/H1 exchanger important for lysosomalacidification, whereas SNX10 plays animportant role in regulating endolysosomaltrafficking. Consistent with the role of thesegenes in apilimod-mediated cell death, CRISPRknockout of TFEB, CLCN7, OSTM1, andSNX10 conferred resistance to apilimod.These results indicate that defects in theacidification of the lysosomal compartmentas well as impaired endolysosomal membranetrafficking are important in apilimod-mediated

Schematic representation of the proposed mechanism of action. By binding to and inhibiting PIKfyve function, apilimod

blocks the formation of PI(3,5)P2 (1). Apilimod mediated-TFEB dephosphorylation promotes TFEB nuclear

translocation and transcription of its target gene, CLCN7 (2). In addition to CLCN7, OSTM1 and SNX10 are

2 other key genes upregulated with apilimod treatment. Both CLCN7 and OSTM1 encode anion exchange

transporters of pivotal importance for lysosome homeostasis, whereas SNX10 plays an important role in regulating

endolysosomal trafficking (3). Apilimod-mediated impairment of lysosomal homeostasis (upregulation of CLCN7,

OSTM1) in the setting of endolysosomal membrane traffic dysfunction (upregulation of SNX10) may induce tumor cell

stress, ultimately leading to lymphoma cell death. LC3, light chain 3; TFEB-p, transcription factor EB-phosphorylated;

TLR9, Toll-like receptor 9; V-ATPase, vacuolar-type H1 ATPase. Professional illustration by Somersault18:24.

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cell death via reduced degradation of thelysosomal cargo (see figure).

Overall, apilimod is an exciting compoundthat produces significant lymphoma celldeath at clinically achievable concentrations.Importantly, its safety and clinical activity arenowbeing evaluated in a phase 1 dose escalationstudy in patients with relapsed/refractoryB-cell NHL (NCT02594384). Regardless,several questions remain that will requirefurther investigation: (1) If this agent is shown tobewell tolerated, is there a role for combinationsof apilimod with chemotherapy in B-cell NHL?It has been shown that autophagy-addictedtumor cells are more susceptible tochemotherapy and radiation when autophagyis inhibited, providing rationale for suchcombination studies. Interestingly, Gayle et alreport significant activity of apilimod in myc-driven DLBCL cell lines. These preliminaryresults cannot be directly translated to DLBCLpatients, of course, but are certainly intriguingconsidering the particularly aggressive natureand the poor prognosis associated withmyc-driven diseases. It is interesting to note thatmyc overexpression via amplificationor translocation induces cytoprotectiveautophagy via the PERK/eIF2a/ATF4pathway in lymphoma, and inhibition ofautophagy in the same model leads to myc-dependent cell death.9 Although further studiesare warranted, the fact that myc-drivenlymphoma could potentially exploit thispathway to escape stressful conditions providesthe rationale for a dual approach with apilimodand chemotherapy specifically in myc-drivenlymphomas. (2) What is the effect of apilimodon immune function? Although the authorsprovide preliminary evidence showing lack ofapilimod cytotoxicity on a variety of normal cellsand tissues, no data are provided on the effectof autophagy inhibition on the function ofimmune cells. The role of autophagy in themaintenance of normal stem cells, in theactivation and proliferation of B andT cells, andin the function of antigen-presenting cells iswelldocumented. For example, tumor-bearingautophagy-deficient mice are unable to mountan effective antitumor response due to theinability to efficiently present tumor antigensand to a defective T-cell–mediated antitumorimmune response.10 Although these aspects canbe preliminarily assessed in the phase 1 clinicaltrial, additional studies will be required toinvestigate how these concepts apply to patientswith cancer treated with autophagy inhibitors

in the presence or absence of immunogenicchemotherapy.Conflict-of-interest disclosure: The author

declares no competing financial interests. n

REFERENCES1. Gayle S, Landrette S, Beeharry N, et al. Identificationof apilimod as a first-in-class PIKfyve kinase inhibitorfor treatment of B-cell non-Hodgkin lymphoma. Blood.2017;129(13):1768-1778.

2. Choi AM, Ryter SW, Levine B. Autophagy in humanhealth and disease. N Engl J Med. 2013;368(7):651-662.

3. Levine B. Cell biology: autophagy and cancer. Nature.2007;446(7137):745-747.

4. Amaravadi RK, Yu D, Lum JJ, et al. Autophagyinhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007;117(2):326-336.

5. Alinari L, Mahoney E, Patton J, et al. FTY720increases CD74 expression and sensitizes mantle celllymphoma cells to milatuzumab-mediated cell death.Blood. 2011;118(26):6893-6903.

6. Vogl DT, Stadtmauer EA, Tan KS, et al. Combinedautophagy and proteasome inhibition: a phase 1 trial of

hydroxychloroquine and bortezomib in patients withrelapsed/refractory myeloma. Autophagy. 2014;10(8):1380-1390.

7. Cai X, Xu Y, Kim YM, Loureiro J, Huang Q.PIKfyve, a class III lipid kinase, is required for TLR-induced type I IFN production via modulation of ATF3.J Immunol. 2014;192(7):3383-3389.

8. Krausz S, Boumans MJ, Gerlag DM, et al. Briefreport: a phase IIa, randomized, double-blind, placebo-controlled trial of apilimod mesylate, an interleukin-12/interleukin-23 inhibitor, in patients with rheumatoidarthritis. Arthritis Rheum. 2012;64(6):1750-1755.

9. Hart LS, Cunningham JT, Datta T, et al. ERstress-mediated autophagy promotes Myc-dependenttransformation and tumor growth. J Clin Invest. 2012;122(12):4621-4634.

10. Michaud M, Martins I, Sukkurwala AQ, et al.Autophagy-dependent anticancer immune responsesinduced by chemotherapeutic agents in mice. Science. 2011;334(6062):1573-1577.

DOI 10.1182/blood-2017-02-764639

© 2017 by The American Society of Hematology

l l l THROMBOSIS AND HEMOSTASIS

Comment on Riedl et al, page 1831

Risking thromboembolism:podoplanin and glioma-----------------------------------------------------------------------------------------------------

Jeffrey I. Zwicker BETH ISRAEL DEACONESS MEDICAL CENTER

In this issue of Blood, Riedl et al evaluate the link between tumor podoplaninexpression and the prothrombotic state inherent to glioma and find thatpodoplanin expression is associated with altered markers of coagulation activationand an increased risk of venous thromboembolism.1

The care of patients with glioma isfrequently influenced by the developmentof venous thromboembolism. After decadesof investigation, the mechanism by whichmalignant brain tumors influence coagulationremains speculative at best. In the 1970s, it wastheorized that thrombosis in brain tumorswas largely the result of spatial disruption

causing “a derangement of normal

hypothalamic control of anticoagulation.”2

Much attention has focused on the role of

tissue factor in mediating the hypercoagulable

state in glioma. Tissue factor is overexpressed

in glioma, but the association between tumor

expression or circulating activity and venous

thromboembolism in glioma is debated.3

Building on emerging data focusing on platelet

activation as central to cancer-associated

thrombosis, Riedl et al now provide evidencethat increased podoplanin expression inglioma cells coincides with the developmentof venous thromboembolism.

Podoplanin is a transmembranesialoglycoprotein expressed in normaltissue, including lymphatic endothelialcells, and is thought to play a critical role inthe embryonic division of lymphatic andvascular systems.4 Aberrant expressionof podoplanin was initially identified ina mouse colonic adenocarcinoma cell line andlater documented in a wide range of tumors,including colon, lung, ovary, testicularseminoma, and brain.5 Interestingly, theinitial description of podoplanin expressionin tumor cells was tied to its potential rolein mediating thrombosis in malignancy.5

1742 BLOOD, 30 MARCH 2017 x VOLUME 129, NUMBER 13

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doi:10.1182/blood-2017-02-7646392017 129: 1740-1742

Lapo Alinari Toward autophagy-targeted therapy in lymphoma

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