Targeting Myddosome Signaling in Waldenstrom's ......Selleck Chemicals. Other reagents were from Sigma-Aldrich Corporation, unless otherwise indicated below. Cell lines and patient

Translational Cancer Mechanisms and Therapy

Targeting Myddosome Signaling inWaldenstr€om's Macroglobulinemia with theInterleukin-1 Receptor-Associated Kinase 1/4Inhibitor R191Haiwen Ni1,2, Fazal Shirazi2, Veerabhadran Baladandayuthapani3,Heather Lin3, Isere Kuiatse2, Hua Wang2, Richard J. Jones2, Zuzana Berkova2,Yasumichi Hitoshi4, Stephen M. Ansell5, Steven P. Treon6, Sheeba K. Thomas2,Hans C. Lee2, Zhiqiang Wang2, R. Eric Davis2, and Robert Z. Orlowski2,7

Abstract

Purpose: Waldenstr€om's macroglobulinemia is an incur-able lymphoproliferative disorder driven by an L265P muta-tion in the myeloid differentiation primary response gene88 (MYD88), which activates downstream NF-kB signalingthrough the Myddosome. As this pathway depends in part onactivity of interleukin-1 receptor-associated kinases (IRAKs)-1and -4, we sought to evaluate the potential of the IRAK1/4inhibitor R191 in preclinical models.

Experimental Design: Patient-derived cell lines and pri-mary samples were used in both in vitro and in vivo experi-ments to model Waldenstr€om's macroglobulinemia and itsresponse to IRAK1/4 inhibitors.

Results:R191 induced a dose- and time-dependent reduc-tion in viability of BCWM.1 and MWCL-1 Waldenstr€om'scell lines, and suppressed activation of IRAK1/4. This wasassociated with cell-cycle arrest at G0–G1, reduced levelsof cyclin-dependent kinases 4 and 6, and induction of

apoptosis in cell lines and primary patient samples. Furtherdownstream, R191 exposure led to reduced activation ofNF-kB, and of protein kinase B/Akt/mammalian target ofrapamycin signaling, whereas expression of a constitutivelyactive Akt mutant induced R191 resistance. Gene expressionprofiling and gene set enrichment analysis revealed a sig-nature consistent with inhibition of c-Myc and activation ofthe endoplasmic reticulum stress response. In both subcu-taneous and systemic murine models of Waldenstr€om's,R191 showed antitumor activity. Finally, the activity ofR191 was enhanced when it was combined with novelchemotherapeutics such as bortezomib, afuresertib, andibrutinib.

Conclusions: Taken together, these data support thetranslation of R191 as an approach to target IRAK1/4 tothe clinic for patients with Waldenstr€om's macroglobuline-mia. Clin Cancer Res; 24(24); 6408–20. �2018 AACR.

IntroductionWaldenstr€om's macroglobulinemia is diagnosed in patients

who have at least 10% bone marrow involvement with lym-

phoplasmacytic lymphoma and a serum monoclonal IgM para-protein (1, 2). Although some patients have asymptomaticdisease which can be followed without treatment, others presentwith, or progress to symptomatic Waldenstr€om's due to bulkydisease, cytopenias, constitutional symptoms, or hyperviscosity,all of which require treatment. Chemotherapy for this diseasehas traditionally been based on the use of various combinationsof drugs, including alkylating agents, anti-CD20 monoclonalantibodies, corticosteroids, nucleoside analogues, and protea-some inhibitors (1, 2). These regimens typically are effective atreducing disease burden and provide good long-term outcomes,but complete responses are rare and Waldenstr€om's is stillconsidered incurable.

Understanding of the molecular pathobiology ofWaldenstr€om's took a huge leap forward with the finding thatthe vast majority of patients harbor an L265P mutation in themyeloid differentiation primary response gene 88 (MYD88;refs. 3, 4). MYD88 is recruited to activated Toll-like receptors(TLR) and interleukin-1 receptor (IL-1R; refs. 5, 6), resulting information of MYD88 homodimers, which then interact withinterleukin-1 receptor-associated kinase 4 (IRAK4) and IRAK2to form the so-called Myddosome (7). Subsequent recruitmentto, and phosphorylation of IRAK1 by the Myddosome is

1Department of Hematology, The Affiliated Hospital of Nanjing University ofTraditional Chinese Medicine, Nanjing, JangSu, China. 2Department of Lympho-ma andMyeloma, TheUniversity of TexasMDAnderson Cancer Center, Houston,Texas. 3Department of Biostatistics, The University of Texas MD AndersonCancer Center, Houston, Texas. 4Rigel, South San Francisco, California. 5Divisionof Hematology, Mayo Clinic, Rochester, Minnesota. 6Dana Farber Cancer Insti-tute, Harvard Medical School, Boston, Massachusetts. 7Department of Experi-mental Therapeutics, The University of Texas MD Anderson Cancer Center,Houston, Texas.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H. Ni and F. Shirazi contributed equally to this work.

Corresponding Author: Robert Z. Orlowski, Department of Lymphoma andMyeloma, The University of Texas MD Anderson Cancer Center, 1515 HolcombeBlvd., Unit 429, Houston, TX 77030. Phone: 713-794-3234; Fax: 713-563-5067;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-3265

�2018 American Association for Cancer Research.

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required for binding of IRAK1 to tumor necrosis factor receptor–associated factor 6 (TRAF6), an E3 ubiquitin ligase that med-iates activation of the inhibitor of NF-kB kinase (IKK). OnceIKK is activated, it phosphorylates the inhibitor of NF-kB (IkB),prompting its ubiquitination and proteasome-mediated deg-radation, freeing NF-kB to translocate to the nucleus andactivate its gene expression program (reviewed in ref. 7) topromote B-cell survival and proliferation (8–15). The L265Pmutation within the MYD88 Toll/interleukin-1 receptor (TIR)homology domain changes its structure and induces receptoractivation-independent Myddosome formation, leading toconstitutive NF-kB activation (16).

In Waldenstr€om's models, MYD88L265P forms complexeswith and activates Bruton's tyrosine kinase (BTK), providinga rationale for the use of tool compound BTK inhibitors,which showed activity against preclinical models (3). Thesefindings set the stage for testing of the BTK inhibitor ibrutinib,whose remarkable clinical activity against relapsed and/orrefractory Waldenstr€om's (17) prompted its regulatoryapproval. Because IRAK1/4 remains an interesting target forthis disease, we set out to evaluate the activity of a novel set ofpotent and specific inhibitors of these kinases (18). Asreported herein, these agents reduced viability and activatedapoptosis in Waldenstr€om's models, inhibited NF-kB andprotein kinase B/Akt signaling, showed efficacy against prima-ry Waldenstr€om's cells and in vivo, and acted synergisticallywith other drugs. Thus, these studies provide a good rationalefor moving these IRAK1/4 inhibitors to the clinic for patientswith Waldenstr€om's macroglobulinemia.

Materials and MethodsReagents

The IRAK1/4 inhibitors R191, R568, and R630 were provid-ed by Rigel Pharmaceuticals, Inc. (18). R191 was profiled in aPanlabs Hit Profiling Screen (30 targets), and gave significant(>50%) inhibition of three targets at 10 mmol/L other thanIRAK1/4: Adenosine A1 (51%), cannabinoid CB1 (97%), andserotonin (5-hydroxltryptamine) 5-HT2B (96%). In follow-upcellular assays at Panlabs, R191 did not show any significant

effect on cannabinoid CB1-induced GTPgS binding in CHO-K1cells, but did antagonize serotonin 5-HT2B-induced inositolmonophosphate (IP1) release. R191 blocked IP1 release withan EC50 of 1.93 mmol/L, which may be due in part to cellcytotoxicity, as R191 caused cytotoxicity in CHO-K1 cells withan EC50 of 8.46 mmol/L, but R191 did not significantly inhibit apanel of related serotonin receptors. The effect of R191 was alsoevaluated on the human potassium channel using humanembryonic kidney (HEK) 293 cells transfected with a humanEther-a-go-go-related gene (hERG), and the maximal inhibitionby R191 of the potassium-selective IKr current was 36.6% at 10mmol/L. With regard to selectivity, R191 was profiled against apanel of 76 kinases representative of the different subfamiliesof the full kinome. In addition to IRAK1 and IRAK4 (IC50 3nmol/L), R191 inhibited the kinase activity of 11 kinases by>80% at 250 nmol/L and two kinases at 50 nmol/L. Theprimary off-target activity of R191 was against Fms-relatedtyrosine kinase (FLT)-3, which was confirmed in cellular assays[Flt3 ligand-induced extracellular signal-regulated kinase-1/2phosphorylation and MV411 (Flt3-internal tandem duplica-tion) proliferation]. In addition, R191 inhibited the cellularactivity of additional targets (TAK1, AXL receptor tyrosinekinase, TANK binding kinase 1, and Bruton's tyrosine kinase)at concentrations of �400 nmol/L or above, at least 20-foldover its activity on IRAK1/4 signaling.

Afuresertib, bortezomib, and ibrutinib were purchased fromSelleck Chemicals. Other reagents were from Sigma-AldrichCorporation, unless otherwise indicated below.

Cell lines and patient samplesThe Waldenstr€om's cell line BCWM.1 was a kind gift from

Dr. Steven Treon (Dana Farber Cancer Institute, Boston, MA).These cells are CD5�/CD10�/CD19þ/CD20þ/CD23þ/CD27�/CD38þ/CD138þ/CD40þ/CD52þ/CD70þ/CD117þ/cIgMþ/cIgG�/cIgA�/ckappa�, and clambdaþ, harbor the MYD88 L265P muta-tion, and express low levels of wild-type CXCR4 (19). MWCL-1Waldenstr€om's cells were generously provided by Dr. StephenAnsell (The Mayo Clinic, Rochester, MN), and are CD3�/CD19þ/CD20þ/CD27þ/CD38þ/CD49Dþ/CD138þ/cIgMþ and ckappaþ.They also harbor the MYD88 L265P mutation and a wild-typeCXCR4, but they have a TP53 missense mutation at exon5 (V143A, GTG>GCG; ref. 19). Cells were propagated inRPMI 1640 media (Thermo Fisher/Life Technologies) supple-mented with 10% FCS (GE Healthcare/HyClone), 2 mmol/LL-glutamine (Thermo Fisher/Invitrogen), 100 U/mL of penicil-lin, and 100 mg/mL of streptomycin (both from Mediatech).Cell line authentication was performed by the MD AndersonCharacterized Cell Line Core Facility using short tandem repeatDNA fingerprinting with the AmpFlSTR Kit (Thermo Fisher/Applied Biosystems).

Three primary samples were obtained from patients undergo-ing bone marrow aspiration after they had provided informedconsent in compliance with the Declaration of Helsinki andaccording to an Institutional Review Board-approved protocol.CD20þ primary cells were purified by positive selection usingmagnetic-activated cell sortingwith CD20þmicrobeads (MiltenyiBiotec).

Peripheral blood mononuclear cells (PBMCs) were isolatedfrom the buffy coats of healthy donors after standard Ficoll-Paquegradient separation, and all the experiments were performed inRPMI 1640 medium.

Translational Relevance

Patients with Waldenstr€om's macroglobulinemia are cur-rently treated with a variety of chemotherapeutics used aloneor in combination, which have been adapted for use based ontheir activity in related disorders, includingmultiplemyelomaand indolent non-Hodgkin lymphomas. The first, and so faronly agent to receive regulatory approval in this disease isthe Bruton's tyrosine kinase ibrutinib, but only a minorityof patients achieve a complete remission with this drug,indicating a need for other rationally targeted therapies. Thisstudy provides evidence that the novel interleukin-1 receptor-associated kinase (IRAK)-1/4 inhibitor R191 has potent activ-ity against preclinical and physiologically relevant models ofthis disease. Because IRAK1/4 is a key signaling intermediatedownstream of the pathobiologic lesion responsible forWaldenstr€om's, this supports the possibility that R191 couldbe an attractive option for translation to the clinic.

IRAK1/4 Inhibitor R191 in Waldenstr€om's Macroglobulinemia

www.aacrjournals.org Clin Cancer Res; 24(24) December 15, 2018 6409




Cell viability assaysThe cells were seeded at 20,000 cells/well on 96-well plates

and treated with vehicle alone, or vehicle with increasingconcentrations of R191 for 24 to 72 hours. Cell viability wastested using the Premix WST-1 Cell Proliferation Assay accord-ing to the manufacturer's instructions (Clontech Laboratories).Conversion of WST-1 to the formazan dye was measured at650 nm using a Victor 3V plate-reader (Perkin Elmer). Dataprovided are representative of at least three independent experi-ments expressed as the mean � SD for samples assayed intriplicate.

Flow cytometryCell-cycle analysis based on DNA content was performed

using propidium iodide (PI) staining. Briefly, cells were col-lected and fixed in 70% cold ethanol in PBS for 2 hours at 4�C.After two subsequent washes with cold PBS, cells were incu-bated with PI staining solution (1� PBS, 100 mg/mL RNAse A,and 40 mg/mL PI) for 30 minutes. For analysis of apoptosis,cells were stained with the Annexin V Apoptosis Detection Kit(BD Biosciences) according to the manufacturer's protocol.Once harvested, cells were resuspended in a binding buffercontaining Annexin V-FITC and PI or TO-PRO-3 and incubatedfor 15 minutes in the dark. Stained cells were analyzed byflow (FACSCalibur; BD Biosciences) using FlowJo software(Tree Star).

Western blottingCells were harvested, washed in PBS, and lysed with buffer

(Cell Signaling Technology) containing protease and phospha-tase inhibitors (Roche Diagnostics). Lysates were sonicated andclarified by centrifugation, and protein concentrations weredetermined using the DC Assay Kit (Bio-Rad). Fifty microgramsof protein was boiled after addition of loading buffer (CellSignaling Technology), separated by gel electrophoresis, trans-ferred onto nitrocellulose, and blocked with Tris-bufferedsaline with 0.01% Tween 20 and 5% nonfat milk prior toincubation with primary and secondary antibodies. Proteinswere visualized using the Enhanced Chemiluminescence Kit(Thermo Fisher/Pierce) according to the manufacturer's instruc-tions and exposed on Hyperfilm-ECL (GE Healthcare Bio-sciences). Protein band intensity was analyzed by densitometryusing the ChemiDoc Touch Imaging System (Bio-Rad) andnormalized to b-actin as a control.

Primary antibodies used included those detecting p44/42MAPK (#9102) and phospho-Thr202/Tyr204 p44/42 MAPK(#4370), Akt (#9272) and phospho-Ser473 Akt (#4058), NF-kBp65 (#8242) and phospho-Ser536 NF-kB p65 (93H1, #3033),phospho-Ser32 IkBa (#2859), IRAK1 (#4359S), IRAK4 (#4363)and phospho-Thr345/Ser346 IRAK4 (#11927), cyclin-dependentkinase (CDK)-4 (#12790) and -6 (#13331), phospho-Ser235/236S6 kinase (#2211), and phospho-Ser21/9 glycogen synthasekinase (GSK)-3a/b (#9327S), all from Cell Signaling Techno-logy. Primary antibodies detecting TRAF6 (ab33915), V-mycavian myelocytomatosis viral oncogene homolog (c-Myc;ab178457), and TATA binding protein (TBP; ab63766) werefrom Abcam. Primary antibodies detecting phospho-Thr209IRAK1 (#SAB4504246), tumor protein p53 inducible nuclearprotein-1 (TP53INP1; #SAB4503641), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4; #SAB1300132),and b-actin (A1978) were from Sigma-Aldrich.

RNA isolation and quantitative real-time PCRTotal RNA extracted using Trizol (Thermo Fisher/Life

Technologies) underwent first-strand cDNA synthesis withthe High-Capacity cDNA Reverse Transcription Kits (AppliedBiosystems). TaqMan Gene Expression Master Mix and probesused to perform quantitative PCR (qPCR) were from AppliedBiosystems, with GAPDH used as internal controls. Reactionswere carried out in triplicate in the ABI PRISM 7900 HT SequenceDetection System (Thermo Fisher/Life Technologies), and relativeamounts of products were determined by the comparative Ctmethod.

Lentiviral transduction and transfection studiesLuciferase was excised from pGL3-basic (Promega) and

ligated into pCDH-CMV-MCS-EF1-copGFP (Systems Bio-scences). Constitutively-active (Akt-DD, T308D/S473D; ref. 20)and dominant-negative Akt (Akt-AAA, K179A/T308A/S473A;ref. 21) mutants from Addgene were cloned into pCDH-CMV-MCS-EF1-puro (Systems Bioscences). Trono vectors (Addgene;ref. 22) expressing shRNA targeting the human c-myc sequencesTGAGACAGATCAGCAACAA (shRNA 1-1) and AACGT-TAGCTTCACCAACA (shRNA 2-5) were used to knockdowncellular c-myc. The Lentiviral vectors were then used for pro-duction of recombinant Lentiviruses by transient transfectionof 293T cells. Briefly, subconfluent 293T cells were cotrans-fected with 20 mg of an expression vector (with or withoutinserted sequences), 15 mg of pAX2, and 5 mg of pMD2G-VSVGby calcium phosphate precipitation. Medium was changedafter 16 hours, and recombinant lentiviruses were harvested24 and 48 hours later. Raw virus supernatants were concen-trated by poly-ethyleneglycol precipitation, and target cellswere transduced in growth medium containing 6 mg/mL poly-brene. Transduced cells were subjected to drug selection, orselected based on GFP expression by flow cytometry at 5 daysposttransduction.

Two IRAK4 loss-of-function mutants (23) were a kind giftfrom Dr. Andrei Medvedev (University of Connecticut HealthCenter, Farmington, CT). The first contains a C-to-T substitu-tion at nucleotide 877 (877–879), creating a premature stopcodon at amino acid 293, whereas the second has an ACdeletion at nucleotides 620–621. They were introduced intothe WT pRK7-IRAK4 vector by site-directed mutagenesis andverified by sequencing. These constructs were cloned intoLentiviral vectors and used for production of BCWM.1-derivedstable cell lines.

Gene expression and reverse phase protein analysesGene expression profiling (GEP) and gene set enrichment

analyses (GSEAs) were performed as described previously (24).For reverse phase protein array (RPPA) analysis, two replicatesof BCWM.1 and MWCL-1 cells were treated with vehicle or1.25 mmol/L R191 for 24 hours. Cells were lysed in RIPA bufferand lysates were centrifuged at 14,000 rpm for 10minutes at4�C. The supernatants were mixed with 4� SDS sample buffer[250 mmol/L Tris, pH 7.4, 2% (w/v) SDS, 25%(v/v) glycerol,and 10%(v/v) 2-mercaptoethanol] and boiled for 5minutes.The RPPA analysis was performed by the MD Anderson RPPACore Facility (http://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core-facilities-and-services/functional-proteomics-rppa-core/index.html) as described previously (25).

Ni et al.

Clin Cancer Res; 24(24) December 15, 2018 Clinical Cancer Research6410



http://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core-facilities-and-services/functional-proteomics-rppa-core/index.html





Animal modelsSix-week-old nonobese diabetic mice with severe combin-

ed immunodeficiency (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) werefrom Jackson Laboratories. BCWM.1 GFPþ/lucþ cells (1 � 107

cells) with Matrigel (BD Biosciences) were injected subcutane-ously. On day 7 post-inoculation, mice were divided intotreatment groups (N ¼ 5 each) to receive intraperitoneal PBSor 20 mg/kg of R191 on days 1 to 5 of three consecutive 7-daycycles. Tumor size/burden was evaluated by three methods:measured by calipers bi weekly, with tumor volume calculatedusing the formula for an ellipsoid sphere [(length�width2)/2];by bioluminescence imaging [after IP injection of 200 mL D-luciferin (15 mg/mL; Caliper Life Sciences) in PBS] usingthe IVIS Imaging System (Caliper Life Sciences) and LivingImage 4.4.SP2 software (Caliper Life Sciences); and by ELISA(Bethyl Laboratories) measuring human IgM levels in samplesfrom tail veins on treatment days 14 and 21. Survivingmice were euthanized and tumor tissue was extracted forfurther analysis.

To develop a model of systemic Waldenstr€om's, BCWM.1GFPþ/lucþ cells (1 � 106) were injected intravenously into thetail veins of mice. On day 3 post-inoculation, mice were dividedinto treatment groups (N ¼ 5 each) and treated as above. Tumorsize/burden was evaluated by bioluminescence imaging, andblood samples were collected for human IgM testing. Animalswere euthanized and bone marrows were harvested for furtheranalysis at the end of the study (day 21 post-inoculation, day 14of treatment). All in vivo modeling was performed under proto-cols approved by the MD Anderson Institutional Animal Careand Use Committee.

Drug synergy calculations and statistical analysesCombination indices (CIs) from drug response data were

calculated using CalcuSyn software (Biosoft). Statistical anal-yses were performed with unpaired t tests in GraphPad, andP values <0.05 were considered statistically significant. The ttest, ANOVA, or their corresponding nonparametric methods(Wilcoxon rank-sum test or Kruskal–Wallis test) were used todetect differences for continuous variables between groups.

ResultsIRAK1/4 inhibition decreased viability in Waldenstr€om'scell lines

We evaluated the potency of a panel of novel IRAK1/4inhibitors which had been identified originally through acell-based screen (18). R191, R568, and R630 all produceda dose-dependent reduction in viability of BCWM.1 andMWCL-1 Waldenstr€om's cells after a 24-hour exposure (Sup-plementary Fig. S1) based on data from tetrazolium dye-basedassays. As the median IC50 was lowest for R191, we selected thisagent for further evaluation. Over the course of a 72-hourexposure, R191 produced a reduction in viability (Fig. 1A),and the time-dependence of its action was demonstrated instudies evaluating 24-, 48-, and 72-hour time points (Fig. 1B).To confirm that R191 inhibited IRAK1 and IRAK4, we analyzedthe expression of the activated/phosphorylated kinases andtotal IRAK1/4 by Western blotting. Notably, R191 significantlydecreased total and phospho-Thr209 IRAK1, and of total andphospho-Thr345/Ser346 IRAK4, in both cell models in a con-centration-dependent manner (Fig. 1C). Finally, R191

decreased levels of the IRAK1-interacting partner TRAF6 in adose-dependent manner (Fig. 1D).

R191 induced cell-cycle arrest and apoptosisTo better understand the mechanisms by which R191 exerted

its effects, we performed cell-cycle analysis of Waldenstr€om'scells treated with R191 for 24 hours. R191 increased thefraction of cells in G0–G1 phase, and decreased the fraction inS phase in both cell lines in a concentration-dependent manner(Fig. 2A; Supplementary Fig. S2A). This apparent G0–G1 cell-cycle arrest suggested that R191 could be modulating levels ofCDK4 and CDK6, and Western blot analysis indeed showeddecreased levels of both in BCWM.1 and MWCL-1 cells(Fig. 2B). We also noticed an increased proportion of cells inthe sub-G0–G1 fractions in R191-treated cells during cell-cycleanalysis, suggesting the activation of programmed cell death.BCWM.1 and MWCL-1 cells were therefore analyzed by flowcytometry after Annexin V-staining, and this assay confirmedthat R191 induced a dose-dependent increase in apoptosis ofthese cells (Fig. 2C, left; Supplementary Fig. S2B), and also in aprimary patient sample (Fig. 2C, right). In order to examine theimpact of R191 on normal cells, we exposed PBMCs to increas-ing R191 concentrations for 24 hours, and then evaluated thelevels of apoptosis. Programmed cell death was activated byR191 in <20% of PBMCs even at 2.5 mmol/L (SupplementaryFig. S3). In contrast, BCWM.1 and MWCL-1 cells were moresensitive, since up to 50% to 60% of cells became apoptotic atthis concentration (Fig. 2C, left), and the same was true forprimary cells, of which 76% were apoptotic at 1.25 mmol/L(Fig. 2C, right). The ability of R191 to activate apoptosis wasconfirmed by Western blot analysis showing an increasedabundance of cleaved and activated caspases-3, -8, and -9, andof cleaved PARP (Fig. 2D, left). Finally, increased levels ofcleaved caspase-3 were seen in primary patient samplesexposed to R191 (Fig. 2D, right).

Inhibition of activation of NF-kB by R191Given the role of Myddosome and IRAK signaling in NF-kB

induction, we next tested NF-kB activity by evaluating the phos-phorylation status of IkBa and the NF-kB subunit p65, as well asnuclear p65 translocation. Western blotting showed decreasedphosphorylation of both IkBa and p65 in BCWM.1 andMWCL-1cells (Fig. 3A). Fractionation studies showed that R191 reducednuclear p65 levels, and enhanced its abundance in the cyto-plasm (Fig. 3B), consistent with inhibition of NF-kB signaling.To further confirm the inhibitory effect of R191, we used lenti-viral transduction to express the degradation-resistant IkBa-super-repressor in BCWM.1 cells and, as expected, this reduceddownstream phospho-p65 levels (Fig. 3C). These super-repressorexpressing cells were more sensitive to R191 treatment (Fig. 3D),confirming the important role of NF-kB signaling as a target forWaldenstr€om's therapy.

IRAK1/4 inhibition suppresses Akt/mTOR signalingActivation of Akt signaling is important to survival and

homing of Waldenstr€om's cells (26), and because it can be adownstream target for NF-kB, we next evaluated the impact ofR191 on protein kinase B/Akt. Judging by the level of activated,phospho-Ser473 Akt by Western blotting, R191 exposuredecreased Akt activation in BCWM.1 (Fig. 4A) and MWCL-1(Fig. 4B) cells. This was associated with decreased activation of






downstream GSK3a/b, as judged by the levels of phospho-Ser21/9 GSK3 (Fig. 4A and B). Similarly, other Akt signalingintermediates showed decreased activation, including PDK1,mTOR, and S6 (Fig. 4A and B), consistent with an inhibitoryeffect on the entire pathway. Moreover, the use of RPPAs toprovide data about the broader proteomic effects of R191confirmed the reduction of activation of several Akt pathwayintermediates (Supplementary Fig. S4A and S4B). To furthersubstantiate the effect of R191 on Akt activation, we nextcompared BCWM.1 cells expressing either a constitutively-active Akt mutant (Akt-DD; T308D/S473D) or a dominant-negative mutant (Akt-AAA; K179A/T308A/S473A). Expressionof the Akt-DD mutant activated downstream signaling as indi-

cated by an increase in phospho-GSK3 and phospho-S6 levels,among others, whereas Akt-AAA reduced these intermediates(Fig. 3C). As expected, BCWM.1 cells expressing the constitu-tively active Akt mutant were more resistant to R191 (Fig. 4D,top), whereas cells expressing dominant negative Akt showedslightly increased sensitivity. Qualitatively similar findings werenoted in MWCL-1 cells expressing the Akt-DD mutant (Fig. 4D,bottom).

Impact of R191 on c-MycTo understand the impact of R191 on gene expression, we

next performed GEP and GSEA, which confirmed inhibition ofAkt/mTOR signaling (Supplementary Table S1; Supplementary

A

B

D

BCWM.1 MWCL-1

R191 (mmol/L) 0 0.3 0.6 1.25 IRAK1

p-IRAK1IRAK4

p-IRAK4β-Ac�n

0 0.3 0.6 1.25

0 0.3 0.6 1.25 R191 (mmol/L) 0 0.3 0.6 1.25

TRAF6

β-Ac�n

Viab

ility

(%)

Viab

ility

(%)

Viab

ility

(%)

Viab

ility

(%)

R191 (mmol/L)

R191 (mmol/L) R191 (mmol/L)

R191 (mmol/L)

* * * * * **

* *

* **

**

**

* *

C

Figure 1.

Viability and IRAK1/4 signaling in R191-treated cell lines. A and B, BCWM.1 andMWCL-1 cells were treated with theindicated concentrations of R191 for 72hours (A), or with selectedconcentrations of R191 for 24, 48, and72 hours (B), and cell viability wasassayed using the tetrazolium reagentWST-1. Data in A and B are expressedas themeans� SD for samples assayedin triplicates, and P values werecalculated for treated cells versusvehicle controls. P values �0.05 areindicated by "�" symbols. C and D,BCWM.1 and MWCL-1 cells weretreated with the indicatedconcentrations of R191, and theexpression and phosphorylation statusof IRAK1 and IRAK4 (C), or theexpression of TRAF6 (D) wereexamined byWestern blotting. b-Actinwas used as a loading control, and arepresentative blot is shown from oneof two independent experiments.

Ni et al.





Fig. S5A). Additional pathway changes of note included inhi-bition of the Wingless-related integration site (Wnt)/b-cateninaxis, and upregulation of CD40 signaling and the endoplasmicreticulum (ER)-stress response (not shown). Interestingly, R191impacted the expression of a c-myc-regulated gene-set (Sup-plementary Fig. S5B), and of genes important for cellularmetabolism, including glycolysis. The effect of R191 on c-mycand PFKFB4 expression was confirmed at the mRNA level byqRT-PCR (Fig. 5A; Supplementary Fig. S6A, respectively), andat the protein level by Western Blotting (Fig. 5B; Supple-mentary Fig. S6B, respectively). To examine if c-myc suppressionwas important to the effects of R191, we compared the viabilityof control BCWM.1 cells and BCWM.1 cells in which c-myc wasdownregulated by shRNAs (Fig. 5C). Downregulation of c-MYC

sensitized BCWM.1 cells to the effects of R191 (Fig. 5D),consistent with this possibility.

As R191 may have off-target effects, we suppressed expres-sion of first IRAK1 and then IRAK4 individually in BCWM.1cells. Decreasing IRAK1 or IRAK4 sensitized these cells to R191(Supplementary Fig. S7A and S7B), as would be expected ifIRAK inhibition is a major mechanism of R191's action sincelower target levels would require less drug to give a similarimpact. Also, we expressed two previously described IRAK4loss-of-function mutants (23), and studied their effect. Expres-sion of both mutants sensitized cells to R191 comparedwith control cells (Fig. 5E) and, at low R191 concentrations,wild-type IRAK4 overexpression protected cells. Also, the loss offunction mutants contributed to inhibition of downstream

Perc

ent o

f liv

e ce

lls

A

B

C

D

Primary cellsPa�ent 1

CDK4

CDK6

β-Ac�n

0 0.3 0.6 1.25

0 0.15 0.3 0.6 1.25

c-Caspase 3

c-Caspase 8

c-Caspase 9

β-Ac�n

c-PARP

β-Ac�n

β-Ac�n

R191 (mmol/L) 0 0.3 0.6 1.25

Perc

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f tot

al c

ells

Apop

tosi

s (%

)R191 (mmol/L) 0 0.15 0.3 0.6 1.25

** **

** ****

* * **

**

**

** ** **

****

R191 (mmol/L) 0 1.25 0 1.25c-

Caspase 3

Primary cellsPa�ent 2 Pa�ent 3

MWCL-1BCWM.1

BCWM.1MWCL-1

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* * * * * *

% G2

% S% G0–G1

Figure 2.

Cell-cycle arrest and apoptosis in R191-treated cell lines and primary cells. A,BCWM.1 and MWCL-1 cells were treatedwith the indicated R191 concentrationsfor 24 hours, and cell-cycle profiles wereassessed by flow cytometry of PI-stained cells. An "�" indicates asignificant (P-value < 0.0001) reductionin the proportion of cells at G0–G1

compared with vehicle-treated controls.B, The expression levels of CDK4 andCDK6 were examined by Westernblotting using b-actin as a loadingcontrol as detailed in the legend to Fig. 1.C, Apoptosis was assessed after 24hours of drug treatment by flowcytometry after Annexin V-staining ofcell lines (left) or of one primary sample(right). Data are expressed as themeans� SD for samples assayed in triplicates,and P values were calculated for cellstreated with R191 vs. untreated controls.Significance levels achieved included"�", indicating a P value <0.009, and"��", indicating a P value <0.0005. D,Apoptosis was assessed by Westernblotting for the expression levels ofcleaved and activated caspases-3, -8,and -9 (designated c-caspase), or ofPARP in cell lines (left), or in two newprimary samples (right).






signaling (Fig. 5F). For example, NF-kB activation as measuredby phospho-p65 levels was reduced to a greater extent by R191in cells with these mutants than in control or wild-type cells.The same was true for phospho-Akt and c-Myc expression,which were decreased to a greater extent by R191 with thedominant-negative constructs than with either approach alone,consistent with the concept that R191 and the mutants had asimilar target.

Induction of an ER stress response by R191GSEA analysis described above had suggested that R191

induced an upregulation of the ER stress response, and wetherefore sought to confirm if this was the case. By usingqRT-PCR, R191 was indeed found to induce increased expres-sion of the 94 kDa glucose-regulated protein (GRP94), acti-vating transcription factor 4 (ATF4), and the spliced variant ofX-box binding protein 1 (XBP1; Supplementary Fig. S8A, upperpanels). Moreover, message for CCAAT-enhancer-binding

protein homologous protein (CHOP) and ER degradationenhancing alpha-mannosidase like protein 1 (EDEM) wereenhanced as well (Supplementary Fig. S8A, lower panels).Because eukaryotic cells often induce autophagy to overcomeER stress (27), we also evaluated the impact of R191 on theautophagic machinery. Increased levels of microtubule-associated protein 1A/1B-light chain 3 form II were seen inBCWM.1, and to some extent in MWCL-1 cells after exposureto R191 (Supplementary Fig. S8B). In addition, levels of p62/Sequestosome 1, which can be degraded upon autophagy pro-cessing (28), were decreased by R191 (Supplementary Fig. S8B).

Antitumor activity of R191To test the activity of R191 against Waldenstr€om's in vivo,

immunodeficient mice were inoculated with BCWM.1 cellssubcutaneously to provide a local model, or intravenouslyto provide a systemic model. Mice bearing subcutaneousxenografts received three R191 cycles starting on day 3

BCWM.1 MWCL-1

0 0.15 0.3 0.6 1.25

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A

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Figure 3.

Inhibition of NF-kB in R191-treated cell lines.BCWM.1 and MWCL-1 cells were treated withindicated R191 concentrations for 24 hours. TheNF-kB activation status was evaluated byWestern blotting of whole cell lysates todetermine the expression and phosphorylationstatus of IkBa and the p65 subunit of NF-kB (A),or of nuclear and cytoplasmic fractions for the p65subunit (B). b-Actin was used as the control fortotal lysates and cytoplasmic fractions, whereasTBP was used for the nuclear fraction. Also,BCWM.1 cells expressing an IkBa super-repressor(S32A/S36A) or vector control sequences weregenerated (C) in which the super-repressorreduced phospho-p65 levels. These cells werethen exposed to the indicatedR191 concentrationsfor 24 hours (D), and cell viabilitywas determined.Data are expressed as the means � SD forsamples assayed in triplicate, and "�" indicatesP values <0.003, whereas "��" indicates P values<0.0001 for the noted comparisons.

Ni et al.





post-inoculation, whereas systemic xenograft-bearing micereceived two cycles starting on day 7 post-inoculation. R191significantly delayed growth of subcutaneous Waldenstr€om'scells based on caliper measures of tumor size (Fig. 6A, left),and prolonged survival of these mice (Fig. 6A, right). Usingwhole-animal in vivo imaging to measure disease burden, R191significantly delayed tumor growth in both the subcutaneous(Fig. 6B, left; Supplementary Fig. S9A) and systemic models(Fig. 6B, right; Supplementary Fig. S9B). This reduction wasparalleled by a reduced level of circulating human IgM in the

serum of mice with subcutaneous (Fig. 6C, left) and systemicdisease (Fig. 6C, right). Notably, there were no significantchanges in the weights of R191-treated mice throughout thein vivo experiments (Supplementary Fig. S8C). As had been thecase in cell line studies, R191 reduced expression of CDK6, andenhanced the levels of cleaved caspase 3 in vivo (Supplemen-tary Fig. S10). Finally, analysis of bone marrows from micewith systemic disease showed decreased levels of GFPþ/CD20þ

tumor cells in the R191-treated mice compared with vehicle-treated mice (Fig. 6D).

A

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1.0 1.0 1.1 0.8 0.71.0 0.9 1.0 0.9 0.8

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R191 (mmol/L)R191 (mmol/L)

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HAAkt

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Akt-DD

0 0.3 0.6 1.25

p-PDK1

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Figure 4.

Inhibition of Akt and mTOR pathways in R191-treated cells. BCWM.1 (A) andMWCL-1 cells (B)were treated with R191 for 24 hours, and theexpression and/or phosphorylation status ofthe indicated proteins was examined byWestern blotting using b-actin as a loadingcontrol. Cell lines were generated thatexpressed either a dominant-negativeAkt-AAA construct or a constitutively-activeAkt-DD construct, which impacteddownstream Akt signaling as would bepredicted (C). BCWM.1 and MWCL-1 cellsexpressing these mutants were treated withR191 for 24 hours, and cell viability wasevaluated (D). Data are expressed as themeans � SD for samples in triplicate, and"�" indicates P values <0.009, whereas "��"indicates P values <0.003, and "��" indicatesP values <0.0001.






BCWM.1

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Figure 5.

Expression of c-Myc in R191-treated cells. The impact of R191 treatment for 24 hours on BCWM.1 and MWCL-1 cells was studied at the mRNA levelusing qRT-PCR (A), and at the protein level by Western blot analysis (B). Data in A are expressed as the means � SD for samples assayed in triplicates,and "�" indicates P values <0.003, whereas "��" indicates P values �0.0003. Expression of c-Myc was suppressed in BCWM.1 cells using twodifferent shRNAs (designated 1-1 and 2-5 in C), and these were then treated with R191 and viability was studies with the WST-1 assay (D) andcompared with a nontargeting (NT) control. Data are expressed as the means � SD for samples assayed in triplicates, and "�" indicates P values �0.0001.Two loss-of-function IRAK4 mutants were expressed in BCWM.1 cells, exposed to the indicated concentrations of R191 for 72 hours, and the viabilitywas then determined and expressed as detailed above, and compared to control cells and cells with wild-type (WT) IRAK4 (E). Western blottingwas then performed to evaluate the impact of these mutants on downstream targets of interest (F).

Ni et al.





Surv

ival

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Figure 6.

Impact of R191 on BCWM.1 xenografts in vivo. Mice were inoculated subcutaneously with 1 � 107 GFPþ/lucþ BCWM.1 cells, or intravenously with 1� 106 BCWM.1GFPþ/lucþ cells. They were then randomized and treated with intraperitoneal injections of R191 or vehicle (Control) with five mice per cohort. Growthof subcutaneous xenografts was measured by calipers and the mean values � SD are shown (A), with "��" indicating P values <0.0001 (left). TheKaplan–Meier method was also used to show survival of the xenograft-bearing mice (right). Survival was defined as the time until the tumors exceeded15 mm in the largest dimension, at which point the animals were euthanized to avoid undue distress. In an independent set of experiments, tumorburden was measured by whole animal in vivo imaging in either a subcutaneous (left) or systemic (right) mouse model at the indicated time-points (B). Themean values � SD are shown, and "��" indicates P values <0.0001. As an added measure of disease burden, the serum levels of human IgM were determinedby ELISA (C) in subcutaneous xenograft-bearing mice at treatment days 14 and 21 (left), and in intravenous xenograft-bearing mice at the end of theexperiment (right). The proportion of GFPþ/CD20þ cells in bone marrows of the systemic (IV) xenograft-bearing mice was evaluated by flow cytometryat the end of the experiment (3 weeks post-inoculation; D). The mean values per group � SD are shown, and "��" indicates P values <0.0001.






Synergy of R191 with other chemotherapeuticsEarlier mechanistic insights into the activity of R191 sug-

gested that combining it with other targeted therapiescould in the future provide attractive options for the clinic.In that the expression of a dominant-negative Akt sensitizedcells to R191, we evaluated the Akt inhibitor afuresertib (29).R191 and afuresertib together enhanced the reduction ofviability in both Waldenstr€om's cell lines (SupplementaryTable S2), with combination indexes well below 1.0, indicat-ing strong synergy. Because BTK is another target in theMyddosome signaling pathway, we tested the ability of ibru-tinib to add to the efficacy of R191, and strongly synergisticeffects were seen between the two in BCMW.1 cells (Supple-mentary Table S2). Because ER stress was induced by R191, wenext combined it with bortezomib, another agent that worksin part through inducing a stress response (30), and synergywas again seen in BCMW.1 cells (Supplementary Table S2).Finally, the alkylating agent bendamustine is one of thestandards of care for initial therapy as part of a combinationwith rituximab for Waldenstr€om's patients. We therefore com-bined bendamustine with R191, and found synergistic effectsat lower bendamustine concentrations, though at higher onesthere was an additive effect, with combination indices ofapproximately 1.0.

DiscussionGenomic studies of Waldenstr€om's macroglobulinemia have

identified a number of commonly recurring mutations in thisdisease, which clinically shares some features of a low-grade B-celllymphoma and a plasma cell dyscrasia. Among these are muta-tions ofCXCR4, which are found in30% to40%of patients, of AT-rich interaction domain1A (ARID1A), whichoccur in 17%, andofCD79B, which can be detected in 8% to 15% of patients (31).However, the most common, and essentially pathognomoniclesion is in MYD88, which is seen in 95% to 97% of patients.Moreover, the downstream effects of this mutation include for-mation of MYD88 homodimers and, in combination with IRAKs,the so-called Myddosome. This produces constitutive NF-kBactivation, and provides downstream proliferative and survivalsignaling to these cells. Validation of the importance of thispathway's role in the pathobiology of this disease was providedby the strong clinical activity of the BTK inhibitor ibrutinib (17),which remains the only agent that has achieved regulatoryapproval for Waldenstr€om's. Indeed, the overall response rate of90.5% was so impressive that this drug was approved for bothnewly diagnosed and more advanced patients even though thestudy enrolled only those with relapsed or refractory disease.However, complete responses are rare, and treatment-emergenttoxicities are not infrequent, including neutropenia in >20%,thrombocytopenia in almost 15%, and epistaxis or atrial fibril-lation in up to 5%. These findings suggest that other therapeutictargets remain of relevance in this disease, and becauseMYD88L265P activates NF-kB through both BTK and IRAK-1/4(32), the latter could clearly be considered in this category.

Based on the rationale above, we evaluated the therapeuticpotential of R191, a newly developed IRAK1/4 inhibitor, inpreclinical Waldenstr€om's models. R191 suppressed the viabilityof, and induced apoptosis in patient-derived cell lines andpatient-derived primary samples while reducing activation ofIRAKs 1 and 4 (Figs. 1 and 2). Moreover, R191 decreased levels

of TRAF6, a crucial component of MYD88L265P–induced IRAK-mediated signaling (33), and subsequent NF-kB activation (34)in cell lines (Figs. 2 and 3). Beyond that, Akt signaling is critical tothe survival and homing of Waldenstr€om's cells (26), and R191suppressed activation of this pathway (Fig. 4), providing furthersupport for its rational use against this disease.

Beyond showing activity against cells grown in culture, R191delayed the growth of, and induced apoptosis in murineWaldenstr€om's models in which tumor cells were grown eitheras a local, subcutaneous mass, or after systemic, intravenousinjection (Fig. 6). Moreover, as would be hoped for from a drugwith potential for clinical application, R191 reduced humanIgM levels in the sera of tumor-bearing mice, and bone marrowinvolvement with Waldenstr€om's cells (Fig. 6). In addition to itsability to induce apoptosis and reduce anti-apoptotic Akt sig-naling, this activity may have been in part due to R191-medi-ated reduction of c-MYC expression (Fig. 5), which correlatedwith cell-cycle arrest and decreased CDK4 and CDK6 levels(Fig. 1). Finally, R191 showed promising activity in combina-tion with agents currently in use against Waldenstr€om's (Sup-plementary Table S2), including afuresertib, bendamustine,and bortezomib, the latter possibly due to an impact on stresspathways (Supplementary Fig. S8). Calcium dysregulation,oxidative damage, and perturbations in posttranslational pro-tein modifications can lead to accumulation of misfolded/unfolded proteins and ER stress. An unfolded protein response(UPR) is triggered to expand the processing capacity of theoverwhelmed ER (35). Production of large quantities of IgMby Waldenstr€om's cells puts a large burden on the proteinprocessing machinery, and makes them susceptible to ER stres-sors. Indeed, proteasome inhibitors were shown previously toinhibit proliferation and to induce apoptosis in Waldenstr€om'smodels (36), and are therefore used against this disease inpatients. Interestingly, there was also strong synergy, as mea-sured by combination indices of 0.251 to 0.496, with ibrutinibitself, supporting the possible use of dual BTK and IRAK1/4inhibitor regimens. Members of the latter category, such asPF-06650833 (37), are currently in phase I and II trials forpatients with inflammatory conditions such as rheumatoidarthritis (NCT02996500), suggesting they could be applied toWaldenstr€om's as well.

Outside of Waldenstr€om's macroglobulinemia, IRAK1/4inhibitors could have other applications. For example, aPhe196Ser mutation of IRAK1 is a common, essential driverfor Kaposi sarcoma herpesvirus lymphoma (38), and theMYD88L265P mutation occurs in almost 30% of activated B-cell like diffuse large B-cell lymphomas (39). Also, IRAK1activation and overexpression have been reported in myelo-dysplastic syndrome and acute myeloid leukemia (40), andmay play a role in melanoma as well (41). Overall, as ourdata suggest that R191 effectively inhibited the BTK-IRAK1/4-NF-kB signaling axis in Waldenstr€om's cells in vitro andin vivo, further studies may be warranted of this agent in theseother malignancies as well.

Disclosure of Potential Conflicts of InterestR.Z. Orlowski is a consultant/advisory board member for BioTheryX,

Bristol-Myers Squibb, Celgene, GSK Biologicals, Janssen Pharmaceutica,Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners,and Takeda Pharmaceuticals North America Inc. No potential conflicts ofinterest were disclosed by the other authors.

Ni et al.





Authors' ContributionsConception and design: H. Ni, F. Shirazi, H. Lin, Y. Hitoshi, S.P. Treon,R.Z. OrlowskiDevelopment of methodology: H. Ni, H. Wang,Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I. Kuiatse, S.M. Ansell, S.K. Thomas, Z. Wang,R.E. Davis, R.Z. OrlowskiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Ni, V. Baladandayuthapani, H. Lin, R.J. Jones,Z. Wang, R.E. Davis, R.Z. OrlowskiWriting, review, and/or revisionof themanuscript: F. Shirazi,H. Lin, I. Kuiatse,R.J. Jones, Z. Berkova, R.Z. OrlowskiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): I. Kuiatse, Z. Berkova, Y. Hitoshi, S.P. TreonStudy supervision: R.Z. OrlowskiOther (designed and performed most of the initial research): H. NiOther (consented patients for primary sample collection and providedhelpful suggestions): H.C. Lee

AcknowledgmentsR.Z. Orlowski, the Florence Maude Thomas Cancer Research Professor,

would like to acknowledge support from the National Cancer Institute (R01CA194264, R01 CA184464, P50 CA142509, U10 CA032102), and theLeukemia & Lymphoma Society (SCOR-12206-17). Core facilities that werekey to these studies, including the Characterized Cell Line Core and the FlowCytometry and Cellular Imaging Core, were supported by the MD AndersonCancer Center Support Grant (P30 CA016672).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedNovember 2, 2017; revised June 18, 2018; accepted August 13, 2018;published first August 20, 2018.

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Ni et al.




2018;24:6408-6420. Published OnlineFirst August 20, 2018.Clin Cancer Res Haiwen Ni, Fazal Shirazi, Veerabhadran Baladandayuthapani, et al. Kinase 1/4 Inhibitor R191Macroglobulinemia with the Interleukin-1 Receptor-Associated Targeting Myddosome Signaling in Waldenström's

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