HCK Is a Survival Determinant Transactivated by Mutated MYD88, and a Direct Target of Ibrutinib

From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only. Regular Article

LYMPHOID NEOPLASIA HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib

Guang Yang,1 Sara J. Buhrlage,2 Li Tan,2 Xia Liu,1 Jie Chen,1 Lian Xu,1 Nicholas Tsakmaklis,1 Jiaji G. Chen,1 Christopher J. Patterson,1 Jennifer R. Brown,3 Jorge J. Castillo,1 Wei Zhang,4 Xiaofeng Zhang,4 Shuai Liu,4 Philip Cohen,5 Zachary R. Hunter,1 Nathanael Gray,2 and Steven P. Treon1

1Department of Medical Oncology, Bing Center for Waldenstrom’s Macroglobulinemia, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA; 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA; 3Department of Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA; 4Department of Chemistry, University of Massachusetts, Boston, MA; and 5Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom

Key Points Activating mutations in MYD88 are present in ∼95% of patients with Waldenstr ¨om macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell • HCK transcription and (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation activation is triggered by of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, mutated MYD88, and is an is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM important determinant of and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell pro-survival signaling. kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). • HCK is also a target of Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/ ibrutinib, and inhibition of AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signal- its kinase activity triggers ing in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK apoptosis in mutated MYD88 inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM cells. and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. (Blood. 2016;127(25):3237-3252) Introduction

Next-generation sequencing has revealed activating MYD88 mutations can also impact ibrutinib response.4 Ibrutinib also shows activity in in several B-cell malignancies. Particularly striking has been the previously treated patients with ABC-DLBCL, particularly among expression of MYD88 mutations in Waldenstrom¨ macroglobulinemia patients with the B-cell receptor (BCR) pathway and MYD88 (WM), wherein 95% to 97% of patients express MYD88L265P,andmore mutations.9 IbrutinibisalsoactiveinotherB-cell malignancies including rarely non-L265P MYD88 mutations.1-4 Up to 30% of patients with chronic lymphocytic leukemia and mantle cell lymphoma. Suppression activated B-cell (ABC) subtype of diffuse large B-cell lymphoma of tonic BCR activity mediated by BTK has been implicated as the (DLBCL) also express activating MYD88 mutations, including mechanism underlying ibrutinib activity in non-WM B-cell diseases. MYD88L265P.5,6 Mutations in MYD88 promote Myddosome self- In addition to BTK, ibrutinib can suppress the activity of several assembly and can trigger NF-kB signaling in the absence of toll-like other kinases including members of the SRC family.10 Hematopoietic receptor or interleukin 1 receptor (IL-1R) signaling through IL-1 receptor cell kinase (HCK) is a member of the SRC family of protein tyrosine associated kinases (IRAK4/IRAK1) or Bruton tyrosine kinase (BTK).5,7 kinases, and one of the most aberrantly upregulated genes in WM Ibrutinib is a pleiotropic kinase inhibitor that is known to target BTK cells.11 In myeloma cells, HCK is activated by IL-6 through the IL-6 co- and is highly active in previously treated WM patients, producing an receptor IL-6ST (GP130).12,13 IL-6 is also a potent growth and survival overall response rate of 91%.8 In WM patients, both major and overall factor in WM, though the functional signiﬁcance of HCK in WM responses to ibrutinib are higher in patients with MYD88 mutations.4 pathogenesis remains unclear.14 We therefore investigated the role of CXCR4 WHIM (warts, hypogammaglobulinemia, infections, and HCKinWM,andimpactofMYD88mutationsandibrutinibonthe myelokathexis) mutations that are present in up to 40% of WM patients transcriptional regulation and activation of this SRC family member.

Submitted January 23, 2016; accepted April 20, 2016. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, May 3, 2016; DOI 10.1182/blood-2016-01-695098. payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. © 2016 by The American Society of Hematology

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 3237 From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3238 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

Figure 1. HCK transcription is driven by mutated A MYD88. (A) HCK transcript levels in MYD88-mutated WM (BCWM.1 and MWCL-1) and ABC DLBCL (TMD-8, 100000 HBL-1, OCI-Ly3, and SU-DHL2) cells, and MYD88 WT 10000 (OCI-Ly7 and OCI-Ly19 GCB DLBCL, Ramos Burkitt, MM1.S, and RPMI-8226 myeloma) cells by qRT-PCR. (B) 1000 HCK transcription using TaqMan Gene Expression Assay in 100 primaryLPCs(CD191) from MYD88 L265P expressing WM 1 2 10 patients, and HD-derived nonmemory (CD19 CD27 )and memory (CD191CD271) B cells. P , .01 for comparison of 1 WM LPC vs either HD non-memory or memory B-cells. The number of healthy donors that were evaluated for nonmemory (open circles) and memory (open triangles) B cells, as well as 0.1

Relative Hck mRNA Levels the number of WM patients that were evaluated for LPC (open diamonds) are shown. (C) Fold change in HCK

TMD-8 HBL-1 Ramos MM1S transcription following lentiviral transduction with vector OCI-Ly3 OCI-Ly7 BCWM.1 MWCL-1 OCI-Ly19 control, Flag-tagged MYD88 L265P or MYD88 WT protein in RPMI-8226 SU-DHL-2 cell lines expressing mutated or WT MYD88. At the bottom of (C), total Flag-tagged MYD88 L265P or WT protein levels are HCK shown for all transduced cell lines to demonstrate relative translation efficiency for MYD88 L265P and WT vectors, as well as GAPDH protein levels as protein loading controls. GAPDH

MYD88 L265P MYD88 WT MYD88 S222R

B 6

2 Relative Hck Expression

0 Non-memory Memory B WM patients B cells (N = 6) cells (N = 6) LPCs (N = 24)

C 10

2 HCK mRNA Fold Change 0

WT WT WT WT Vector L265P Vector L265P Vector L265P Vector L265P MYD88-Flag

GAPDH

BCWM.1 MWCL-1 OCI-Ly19 Ramos

MYD88-L265P MYD88-WT From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3239

AB 1000000 100000 100000 10000 10000 1000 1000 100 100 10 10

Relative IL-6 1 mRNA Levels 1 mRNA Levels Relative IL-6R 0.1 0.1

TMD8 HBL-1 BCWM MWCL Ramos MM1S BCWM MWCL TMD8 HBL-1 Ramos MM1S OCI-Ly3OCI-Ly7 OCI-Ly3OCI-Ly7 OCI-Ly19 SU-DHL-2 OCI-Ly19 RPMI-8226 RPMI-8226 SU-DHL-2 MYD88 L265PMYD88 WT MYD88 MYD88 L265PMYD88 WT MYD88 S222R S222R

CD 10 10

8 8

6 6

4 4

2 2 Relative IL-6 Expression

0 Relative IL-6R Expression 0 Non-memory Memory B WM patients Non-memory Memory B WM patients B cells (N = 6) cells (N = 6) LPCs (N = 24) B cells (N = 6) cells (N = 6) LPCs (N = 24)

EF 10 10

8 8

6 6

4 4

2 2 IL-6 mRNA Fold Change

0 IL-6R mRNA Fold Change 0

WT WT WT WT WT WT WT WT Vector L265P Vector L265P Vector L265P Vector L265P Vector L265P Vector L265P Vector L265P Vector L265P BCWM.1 MWCL-1 OCI-Ly19 Ramos BCWM.1 MWCL-1 OCI-Ly19 Ramos

MYD88 L265P MYD88 WT MYD88 L265P MYD88 WT

Figure 2. IL-6 but not IL-6R transcription is induced by mutated MYD88. Expression of IL-6 (A) and IL-6R (B) transcripts using TaqMan Gene Expression Assays in MYD88-mutated and MYD88 WT cell lines. The number of healthy donors that were evaluated for nonmemory (open circles) and memory (open triangles) B cells, as well as the number of WM patients that were evaluated for LPC (open diamonds) are shown. IL-6 (C) and IL-6R (D) transcript levels were also determined by TaqMan Gene Expression Assays in primary LPCs (CD191) from MYD88 L265P expressing WM patients, and HD-derived nonmemory (CD191CD272) and memory (CD191CD271) B cells. P , .01 for comparison of IL-6 or IL-6R transcript levels in WM LPC vs either HD nonmemory or memory B cells. IL-6 (E) and IL-6R (F) transcription following over-expression of MYD88 L265P or WT protein in cell lines expressing mutated or WT MYD88. Total Flag-tagged MYD88 L265P or WT protein, as well as GAPDH protein levels determined by immunoblotting are shown for all transduced cell lines in Figure 1C.

mutated gatekeeper residue (HCKT333M) at amino acid position 333 (338 based on 15 WT Materials and methods c-SRC numbering) was accomplished by lentiviral transduction of HCK or HCKT333M (r. 1235C.T in NM_002110.3) sequences in a pLVX-EF1a-IRES- Primary cells and cell lines Puro Vector (Clontech Laboratories, Mountain View, CA). Knockdown of HCK or IL-6ST (GP130) was performed using an inducible lentiviral short hairpin RNA 1 Primary WM cells (CD19 ) were isolated from bone marrow (BM) aspirates, (shRNA) expression vector pLKO-Tet-On containing a tetracycline-regulated and peripheral blood mononuclear cells were collected from healthy donors expression cassette (Addgene, Cambridge, MA). Following lentiviral transduction (HDs) after informed written consent was obtained. Primary WM lymphoplas- of BCWM.1 and MWCL-1 cells, stable cell lines were selected with 0.5 to macytic cells (LPCs) and cell lines were genotyped for MYD88 and CXCR4 ;1.0 mg/mL puromycin in a tetracycline-free medium. For the induced knockdown mutations as previously described.2,4 MYD88L265P BCWM.1 and MWCL-1 of HCK or IL-6ST, tetracycline (0.8 mg/mL) was added to culture media. For HCK WM cells; TMD-8, HBL-1, and OCI-Ly3 and MYD88S222R SU-DHL2 knockdown, lentiviral vectors were designed to target 59-GCTGTGATTTG ABC DLBCL cells; and MYD88 wild-type (WT) (MYD88WT)OCI-Ly7and GAAGGGAA-39 (HCK shRNA1) and 59-GGATAGCGAGACCACTAAA-39 OCI-Ly19 germinal center B-cell (GCB) DLBCL cells; Ramos Burkitt cells; (HCK shRNA2). IL-6ST knockdown targeted 59-GGAGCAATATACTAT and RPMI-8226 and MM.1S myeloma cells were used. CATA-39 (IL-6ST shRNA1) and 59-GGAACTGTCTAGTATCTTA-39 (IL-6ST shRNA2). A scrambled shRNA vector was used for control purposes. Lentiviral transduction experiments Cell viability assessments MYD88WT or MYD88L265P proteins were over-expressed in BCWM.1 and MWCL-1 cells following lentiviral transduction as previously described.7 The Apoptosis analysis was performed using Annexin V/propidium iodide (PI) over-expression of proteins coding for WT HCK (HCKWT) or HCK harboring a staining with the Apoptosis Detection Kit I (BD Pharmingen, San Jose, CA). From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3240 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

A MYD88 WT MYD88 L265P

100 100 100 100 MFI: MFI: 80 80 760 80 80 1399

60 60 60 60

40 40 40 40 OCI-Ly7 BCWM.1 0.14 19.6% 0 72.0% 20 20 20 20

0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105

100 100 100 100 MFI: MFI: 80 80 736 80 80 1584

60 60 60 60

40 40 40 40 MWCL-1 OCI-Ly19 0 20.9% 0 88.1% 20 20 20 20

0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105

100 100 100 100 MFI: MFI: 80 80 746 80 80 2005

60 60 60 60

40 40 TMD-8 40 40 0.012 96.4% 0 29.9% 20 20 20 20

0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105

100 100 100 100 MFI: MFI: 80 80 932 80 80 6459

60 60 60 60

40 40 40 40 OCI-Ly3 0.058 90.3% RPMI-8226 Ramos 0 42.9% 20 20 20 20

0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105

Control Ab p-HCK Control Ab p-HCK

Figure 3. HCK is hyperactive in MYD88-mutated cells, and its activation is triggered by IL-6. (A) Results of Phosflow analysis for phosphorylated (p) HCK (pHCKTyr411)in MYD88-mutated WM (BCWM.1 and MWCL-1) and ABC DLBCL (TMD-8 and OCI-Ly3) cells; MYD88 WT GCB DLBCL (OCI-Ly7 and OCI-Ly19), Ramos Burkitt, and RPMI-8226 myeloma cells. (B) Results of Phosflow analysis for pHCKTyr411 in primary LPCs (CD201) from 3 representative MYD88 L265P expressing WM patients, as well as nonmemory (CD201CD272) and memory (CD201CD271) B cells from HDs. Percentage of cells gating for pHCKTyr411 expression are shown in (A-B). Histogram depicts the results of pHCKTyr411 expression in LPC derived from 20 WM patients, as well as nonmemory and memory B cells from 5 HDs. P , .01 for comparison of pHCK expression in WM LPC vs either HD nonmemory or memory B cells. The number of WM patients that were evaluated for LPC (open circles), as well as the number of healthy donors that were evaluated for nonmemory (open diamonds) and memory (open diamonds) B cells are shown. (C) pHCKTyr411 expression in the presence or absence of IL-6 (1 ng/mL) for 5 minutes in MYD88- mutated (BCWM.1, MWCL-1, TMD-8, HBL-1, and OCI-Ly3) and MYD88 WT (OCI-Ly7, OCI-Ly19, Ramos, and RPMI 8226) (top) cell lines, as well as primary WM LPCs. Peak pHCKTyr411 activation was determined by Phosflow analysis after 3 MYD88 mutated cell lines were cultured with IL-6 (1 ng/mL) for 2, 5, and 15 minutes (bottom). *P , .05; **P , .01; ***P , .001 vs untreated controls. (D) Phosflow analysis of pHCKTyr411 in BCWM.1 cells transduced with scrambled control vector or IL-6ST knockdown vector (shRNA2) in the presence or absence of 1 ng/mL of IL-6. Ab, antibody; MFI, mean fluorescence intensity; PBMCs, peripheral blood mononuclear cells; SSC, side scatter.

Cells (1 3 106/well) were treated in 24-well plates for 18 hours with inhibitors or were cultured with tetracycline, and aliquoted every other day on days 1 to 11. corresponding controls. A minimum of 10 000 events were acquired using a BD AlamarBlue solution (1/10 total volume) was added to cells and incubated for FACSCanto II ﬂow cytometer, and analyzed with BD FACS DIVA software. 2 hours. Aliquoted plates were read in a SpectraMax M3 Plate Reader (Molecular For WM patient cells, BM mononuclear cells (2 3 106/well) were treated with Devices, Sunnyvale, CA). Relative cell survival was calculated as a percentage of inhibitors, and CD19–APC-cy7 antibody (BD Pharmingen) was used with ﬂuorescence relative to scrambled control. The CellTiter-Glo Luminescent Cell Annexin V antibody to analyze WM cell apoptosis. AlamarBlue cell viability Viability Assay (Promega, Madison WI) was used to assess the dose-response of assay (Life Technologies, Carlsbad, CA) was used to assess cell death following inhibitors. Cells were seeded into 384-well plates with the EL406 Combination inducible HCK knockdown. For these experiments, transduced cells (1 3 105/mL) Washer Dispenser (BioTek Instruments, Inc.) and inhibitors injected into the cells From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3241

B CD20+ Lymphocytes 5 100 100 10 MFI: 80 80 2538 104 60 60 103 40 40 97.3% 102 0 Patient 1 20 20 0 CD20+ -102 0 0 0103 104 105 0103 104 105 0103 104 105

5 100 100 10 MFI: 80 80 1522 104 60 60 3

SSC 10 40 40 0.041 85.1% 0 CD20+ Patient 2 20 20 -103 0 0 0103 104 105 0103 104 105 0103 104 105

5 100 100 10 MFI: 80 80 1584 104 60 60 3 10 40 40 0 83.1% 0 Patient 3 CD20+ 20 20 -103 0 0 0103 104 105 0103 104 105 0103 104 105 CD20-APC-cy7 Control Ab p-HCK Healthy donor CD20+CD27- CD20+CD27+ lymphocytes 5 100 100 100 10 MFI: 100 MFI: 80 80 222 80 320 104 80 60 60 60 60 103 40 40 40 HD-1 0 7.93% 40 2 8.23% 10 20 20 0 20 0 20 -102 0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105 0103 104 105

105 100 100 100 100 MFI: MFI: 80 80 80 80 104 314 421 60 60 60 60 103 40 40 40 40 HD-2 11.5% 2 0 10 20 0 20 9.92% 0 20 20 2 -10 0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105 0103 104 105 CD27-Alexa Fluor 488 5 100 100 100 100 10 MFI: MFI: 80 80 382 80 80 371 104 60 60 60 60 40 40 40 40 HD-3 0 0.011 8.63% 0 1.73% 20 20 20 20 4 -10 0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105 0103 104 105

CD20-APC-cy7 Control Ab p-Hck Control Ab p-Hck

pHCK expression

100

20 p-Hck positive cells (%)

0 WM-BM-LPCs HD-PBMCs_ HD-PBMCs_ CD20+ CD20+CD27- CD20+CD27+

Figure 3. (Continued). From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3242 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

C 10 Untreated * ** IL-6_5min 8 *** ***

6 *

4 Relative MFI of pHck 2

0 BCWM.1 MWCL-1 TMD-8 HBL-1 OCI-Ly3 OCI-Ly7 OCI-Ly19 Ramos RPMI-8226

10 Untreated ** *** ** 2 min 8 * * 5 min * 6 15 min

2 Relative MFI of pHck

0 BCWM.1 MWCL-1 TMD-8

Untreated + IL-6 100 100 100 MFI: MFI: 80 80 758 80 857 60 60 60 40 40 40

WM Patient 1 20 0 20 44.0% 20 61.4% 0 0 0 0103 104 105 0103 104 105 0103 104 105

100 100 100 MFI: MFI: 80 80 556 80 725 60 60 60 40 40 40 0.060 44.1% 66.3%

WM Patient 2 20 20 20 0 0 0 0103 104 105 0103 104 105 0103 104 105

100 100 100 MFI: MFI: 80 80 781 80 998 60 60 60 40 40 40 0.023 43.7% 64.7%

WM Patient 3 20 20 20 0 0 0 0103 104 105 0103 104 105 0103 104 105 Control Ab p-Hck

Figure 3. (Continued). From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3243

D Scrambled control sh-IL6ST-2 100 100 MFI: MFI: 80 80 536 80 429 60 60 60 Scrambled control sh-IL6ST-1sh-IL6ST-2 40 pHck 40 40 0.021 57.4% 38.0% 20 20 20 IL6ST 0 0 0 0103 104 105 0103 104 105 0103 104 105 GAPDH Control Ab p-Hck

Scrambled control sh-IL6ST-2 Untreated + IL-6 Untreated + IL-6 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 445 80 548 80 433 80 435 60 60 60 60 60 40 40 40 40 40 0.23 41.3% 57.1% 39.1% 40.0% 20 20 20 20 20 0 0 0 0 0 0103 104 105 0103 104 105 0103 104 105 0103 104 105 0103 104 105 Control Ab p-Hck

Figure 3. (Continued). culture media with the JANUS Automated Workstation (PerkinElmer Immunoblotting Inc., Waltham, MA). Cells were treated with serial diluted inhibitors (20 to ;0.0006 mM) for 72 hours at 37°C. Luminescent measurement Immunoblotting was performed following gene over-expression, knockdown, was performed using the 2104 EnVision Multilabel Reader (PerkinElmer or kinase pull-down with biotinylated probes using antibodies for HCK, Inc.). AKT-pT308, AKT, extracellular signal-regulated kinase 1/2 (ERK1/2)-pT202/ pY204, ERK1/2, PLCg1-pY783, PLCg2-pY1217, PLCg2, BTK-pY223, BTK, IRAK4-pT345/S346, IRAK4, SRC, LYN (Cell Signaling Technologies, Danvers, MA), PI3K-p85b-pY464 (LifeSpan Biosciences, Seattle, WA), and Reverse transcription polymerase chain reaction (RT-PCR) PI3K-p85b,PLCg1, IL-6ST (Santa Cruz Biotechnology, Dallas, TX) in primary and quantitative-PCR WM cells, and cell lines. Staining with glyceraldehyde-3-phosphate dehydro- Total RNA were isolated using AllPrep DNA/RNA Mini Kit (QIAGEN), genase (GAPDH) antibodies was used for determination of protein loading and cDNA synthesized by SuperScript III First-Strand Synthesis SuperMix (Santa Cruz Biotechnology). (Life Technologies). Quantitative detection of messenger RNA levels for HCK, IL-6 and IL-6R was performed using TaqMan Gene Expression Assays with TaqMan Gene Expression Master Mix per manufacturer’s Ibrutinib probe assay and kinase active-site inhibition assay instructions using the ABI Prism 7500 Sequence Detection System For ibrutinib probe assay, BCWM.1, MWCL-1, or TMD-8 cells (2 3 107)were (Applied Biosystems). HCK transcription was also assessed two hours treated with ibrutinib-biotin or CC-292–biotin (2 mM) for 1 hour. Cells were then following incubation with either IL-6 (1-10 ng/mL) or IL-6 blocking washed with phosphate-buffered saline twice, and lysed with co-IP buffer antibody (1-10 mg/mL) (both from R&D Systems, Minneapolis, MN) in (Invitrogen, Grand Island, NY). Protein (2 mg) from lysed cells was then BCWM.1 and MWCL-1 cells by quantitative RT-PCR (qRT-PCR). incubated with 50 mL of Pierce Streptavidin Magnetic Beads (Thermo Fisher Scientific, Cambridge, MA) at 4°C for 1 hour, then washed with Tris-buffered saline with Tween 20 (0.1%) thrice, and proteins eluted with sodium dodecyl Phosflow analysis sulfate–polyacrylamide gel electrophoresis sample buffer. For kinase active-site inhibition assay, BCWM.1 cells (2 3 107) were pre-treated with dimethyl Phosflow analysis was performed to delineate HCK phosphorylation. Cells sulfoxide (DMSO), ibrutinib, CC-292, or A419259 (MedChem Express, were fixed with BD Phosflow Fix Buffer I at 37°C for 10 minutes. Cells were Monmouth Junction, NJ) at various concentrations for 1 hour. Cells then were then centrifuged (300g for 5 minutes) and washed twice with Phosflow lysed and kinases were pulled down with adenosine triphosphate (ATP)-biotin Perm/Wash Buffer I (BD Pharmingen). Cells were then stained with HCK using the Pierce Kinase Enrichment Kit (Thermo Fisher Scientific) per the (pTyr411) antibody (Abcam, Cambridge, MA) alone (for cell lines) or with manufacturer’s instructions. Kinases were eluted with sodium dodecyl sulfate– anti-CD20–APC-Cy7 (BD Pharmingen) for primary WM cells. Following polyacrylamide gel electrophoresis sample buffer and detected by western blot. staining, cells were incubated in the dark for 30 minutes at room temperature, then washed thrice with BD Phosflow Perm/Wash Buffer I, followed by anti-rabbit immunoglobulin G DyLight-649 secondary antibody Docking study (Abcam), and incubated for an additional 20 minutes. Cells were then washed twice with BD Phosflow Perm/Wash Buffer I and flow analysis was performed The docking studies were performed using AutoDockTools 1.56,16 AutoDock using a BD FACSCanto II Flow Cytometer. VINA,17 and Open Babel18 software. The lowest calculated Gibbs energy (DG) From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3244 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

A BCWM.1 MWCL-1 TMD-8 HBL-1

Control shRNA1 shRNA2 Control shRNA1 shRNA2 Control shRNA1 shRNA2 Control shRNA1 shRNA2

HCK

GAPDH

B BCWM.1 TMD-8 120 120 Control Vector Control Vector 100 HCK shRNA1 100 HCK shRNA1 80 HCK shRNA2 80 HCK shRNA2 60 60 40 40 20 20 Relative survival (%) Relative survival (%) 0 0 0246810 0246810 Days Days

HBL-1 120 MWCL-1 120 Control Vector Control Vector 100 HCK shRNA1 100 HCK shRNA1 80 HCK shRNA2 80 HCK shRNA2 60 60 40 40 20 20 Relative survival (%) 0 Relative survival (%) 0 0246810 0246810 Days Days

Figure 4. HCK is a determinant of survival in MYD88-mutated cells. (A) HCK protein levels determined by western blot analysis, and (B) survival determined by AlamarBlue cell viability assay over a 9-day evaluation period in MYD88-mutated BCWM.1 and MWCL-1 WM cells (left), and TMD-8 and HBL-1 ABC DLBCL cells (right) following transduction with inducible HCK knockdown (shRNA1 and shRNA2) or scrambled control vectors. Mean of 2 independent experiments is depicted for time points. of the predicted binding modes indicates stronger binding affinity, and binding that carry the MYD88S222R activating mutation. Western blot analysis modes with DGlowerthan210 kcal are highly probable to be true. confirmed enhanced HCK protein expression in MYD88-mutated cell lines (Figure 1A). We next investigated the messenger RNA levels of Statistical analysis HCK in MYD88L265P genotyped CD19-sorted primary WM cells using a The statistical significance of differences was analyzed using one-way analysis of TaqMan Gene Expression Assay. We compared HCK expression levels variance with Tukey’s multiple comparisons test by Prism software. Differences to both sorted HD-derived nonmemory (CD191CD272)andmemory fi , 1 1 were considered signi cant when P .05. (CD19 CD27 ) B cells, given that the later represents the B-cell population from where most cases of WM are likely derived.19,20 The HCK transcript was elevated in MYD88L265P WM cells vs either HD nonmemory or memory B cells (Figure 1B). CXCR4 mutation status Results did not impact HCK transcription in primary WM cells (P 5 .90 for HCK transcription is driven by mutated MYD88 CXCR4 WT vs WHIM-mutated patients). To clarify if mutated MYD88 was responsible for enhanced HCK expression, we over- To clarify if HCK expression was aberrant in MYD88-mutated cells, we expressed MYD88L265P or MYD88WT protein in MYD88L265P first assessed HCK transcription in MYD88-mutated WM and DLBCL BCWM.1 and MWCL-1 cells, and MYD88WT OCI-Ly9 and Ramos cell lines by TaqMan Gene Expression Assay. The results showed cells, and assessed for differences in HCK transcription. By western blot that HCK was markedly transcribed in MYD88L265P expressing WM analysis, similar levels of exogenous MYD88 protein were detectable in (BCWM.1 and MWCL-1) and DLBCL (TMD-8, HBL-1, and OCI-Ly3) MYD88L265P or MYD88WT transduced cells (Figure 1C). The results of cells, but absent or at very low levels in MYD88WT (OCI-Ly7, OCI-Ly19, these studies showed significantly higher levels of HCK transcript in all Ramos, MM1.S, and RPMI-8226) cells by qRT-PCR (Figure 1A). 4 cell lines following over-expression of MYD88L265P vs MYD88WT Expression of the HCK transcript was also enhanced in SU-DHL2 cells protein (Figure 1C). From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3245

A 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 80 80 80 1057 1295 558 458 60 60 60 60 60

40 40 40 40 40 Control 67.0% 55.8% BCWM.1 0 56.3% 43.2% 20 20 20 20 20

0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105

100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 80 80 80 1480 2148 763 541 60 60 60 60 60

HCK 40 40 40 40 40 0.022 88.0% 75.8% BCWM.1 74.0% 58.4% 20 20 20 20 20

0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105

Control Ab p-HCK p-BTK p-AKT p-ERK

100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 80 80 80 660 709 487 421 60 60 60 60 60

40 40 40 40 40 Control 56.8% 56.8% 47.5% MWCL-1 0 37.5% 20 20 20 20 20

0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105

100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 80 80 80 1024 1345 706 489 60 60 60 60 60

HCK 40 40 40 40 40 81.9% 88.6% 71.1% MWCL-1 0 49.7% 20 20 20 20 20

0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105

Control Ab p-HCK p-BTK p-AKT p-ERK

Figure 5. HCK triggers pro-survival signaling in MYD88-mutated WM cells. (A) Phosflow analysis for pHCKTyr411, pBTK, pAKT, and pERK in scramble control vector (top) or HCK transduced MYD88 mutated BCWM.1 and MWCL-1 cells (bottom). (B) Impact of HCK over-expression or (C) HCK knockdown on PI3K/AKT (PIK3R2 and AKT), MAPK (PLCg1 and ERK1/2), BTK (BTK and PLCg2), and IRAK4 signaling in BCWM.1 and MWCL-1 cells using antibodies to detect phospho-specific and total protein expression by immunoblotting. GAPDH protein levels were determined for control purposes in all experiments.

IL-6 but not IL-6R transcription is induced by mutated MYD88 (BCWM.1 and MWCL-1) and MYD88WT (OCI-Ly9 and Ramos) cells. Conversely, over-expression of MYD88WT protein had little or no im- Because previous work showed that HCK was activated by IL-6 via pact on IL-6 or IL-6R transcription. IL-6R/IL-6ST, we investigated the regulatory role of mutated MYD88 on IL-6 and IL-6R expression.12,13 By qRT-PCR, IL-6 transcription was markedly higher in MYD88L265P expressing WM (BCWM.1 and HCK is hyperactive in MYD88-mutated cells, and its activation MWCL-1) and DLBCL (TMD-8, HBL-1, and OCI-Ly3), as well as but not transcription is triggered by IL-6 S222R WT MYD88 expressing SU-DHL2 cells vs MYD88 (OCI-Ly7, Given the above findings, we next investigated the activation state of OCI-Ly19, Ramos, MM1.S, and RPMI-8226) cells (Figure 2A). HCK in MYD88-mutated WM and DLBCL cell lines, and primary Similarly, IL-6R transcription was increased in MYD88-mutated cell WM cells. By Phosflow analysis, HCK showed consistently high WT lines. Among MYD88 malignant B cells, IL-6R transcription was phosphorylation levels at the Tyr411 activation site in mutated MYD88 WT low or absent, although it was highly expressed in MYD88 malignant vs WT MYD88 cells (Figure 3A). Significantly higher levels of HCK plasma cells (Figure 2B). By TaqMan Gene Expression Assay, higher Tyr411 phosphorylation were also observed in primary WM patient IL-6 (Figure 2C) and IL-6R (Figure 2D) transcription were found in samples vs HD non-memory and memory B cells (Figure 3B). 1 2 MYD88L265P WM samples vs HD non-memory (CD19 CD27 )and CXCR4 mutation status was available for 18 of 20 primary WM 1 1 memory (CD19 CD27 ) B cells. Given these findings, we next sought patient samples, and did not impact HCK Tyr411 phosphorylation to clarify if MYD88L265P was a driver for IL-6 and IL-6R transcription. (P 5 .65 for CXCR4 WT vs WHIM-mutated patients). Treatment of Over-expression of the MYD88L265P protein induced marked IL-6 mutated MYD88 WM and DLBCL cell lines and primary WM LPCs (Figure 2E), but not IL-6R (Figure 2F) transcription in MYD88L265P with IL-6 augmented HCK activation, with a peak induction of From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3246 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

Figure 5. (Continued). BC

BCWM.1 MWCL-1 BCWM.1 MWCL-1

VectorHCK Vector HCK ControlshRNA-1shRNA-2 ControlshRNA-1shRNA-2 Hck Hck Overexpression Knockdown

p-BTK p-BTK total BTK total BTK

p-PLCγ2 p-PLCγ2 total PLCγ2 total PLCγ2

p-AKT p-AKT total AKT total AKT

p-PIK3R2 p-PIK3R2 total PIK3R2 total PIK3R2

p-ERK1/2 p-ERK1/2 total ERK1/2 total ERK1/2

p-PLCγ1 p-PLCγ1 total PLCγ1 total PLCγ1

p-IRAK4 p-IRAK4 total IRAK4 total IRAK4

GAPDH GAPDH

Tyr411 phosphorylation at 5 minutes following IL-6 administration in regulator for their signaling.21-24 Transduction of HCK in IL-6– mutated MYD88 cells (Figure 3C). In contrast, little or no effect on producing BCWM.1 and MWCL-1 cells led to higher HCK protein HCK Tyr411 phosphorylation was observed following IL-6 stimu- levels by western blot analysis and detection of activated HCK (Tyr411) lation in MYD88WT cells. Knockdown of IL-6ST attenuated HCK by Phosﬂow analysis (Figure 5A-B). Transduction of HCK in BCWM.1 activation in the presence or absence of IL-6, whereas transduction and MWCL-1 cells also triggered PI3K/AKT (pPIK3R2 and pAKT), with a control vector had little or no impact on HCK activation MAPK (pPLCg1 and pERK1/2), and BTK (pBTK and pPLCg2) in MYD88-mutated BCWM.1 cells (Figure 3D). Lastly, HCK signaling (Figure 5B), whereas knockdown of HCK in BCWM.1 and transcription was not signiﬁcantly altered following incubation with MWCL-1 cells showed a reciprocal pattern, with diminished PI3K/AKT, either IL-6 (1-10 ng/mL) or an IL-6 blocking antibody (1-10 mg/mL) MAPK, and BTK signaling (Figure 5C). IRAK4 activation was not for 2 hours in BCWM.1 and MWCL-1 cells using RT-PCR (data not impacted by either HCK over-expression or knockdown. Total shown). protein levels for these signaling molecules and GAPDH remained unchanged in these experiments. HCK is a determinant of survival in MYD88-mutated cells

We next evaluated the impact of HCK expression on survival in Ibrutinib binds to the ATP-binding pocket of HCK and blocks MYD88-mutatedBCWM.1andMWCL-1WM,andTMD-8and ATP binding HBL-1 ABC DLBCL cells. Successful knockdown of HCK was Kinase selectivity profiling has established that, in addition to being accomplished by lentiviral transduction using inducible shRNA vectors a covalent inhibitor of BTK, ibrutinib is also a potent noncovalent (Figure 4A). The results of these studies showed that knockdown of inhibitor of several SRC family members including HCK, YES, HCK in all 4 MYD88-mutated cell lines by either of the two shRNA and LCK.10 We focused our attention on investigating the potential vectors resulted in a sustained reduction in tumor cell viability over the functional significance of HCK to ibrutinib’s pharmacology due to its 9-day evaluation period in contrast to cells transduced with a control structural similarity with A419259, an HCK inhibitor with established vector (Figure 4B). activity inmurine tumor models.15 Indeed, both ibrutinib and A419259 were developed based on the 4-amino-5,7-substituted pyrrolopyrimidine HCK triggers pro-survival signaling in MYD88-mutated WM cells scaffold of the classical SRC-family inhibitor: PP1.25 To create a In view of the above findings, we next interrogated HCK-dependent model for how ibrutinib might bind to HCK, we performed molecular survival signaling in MYD88-mutated WM cells. We focused our docking studies of ibrutinib into the co-crystal structure of HCK with efforts on PI3K/AKT, mitogen-activated protein kinase (MAPK), and A419259 (RK-20449) (PDB 3VS3)26 (Figure 6A). As expected, the BTK signaling pathways given their established importance in WM docking study predicts that ibrutinib recognizes the ATP-binding survival, as well as previous work implicating HCK as an upstream pocket of HCK with an almost identical pose as A419259 with a From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3247

Figure 6. Ibrutinib binds to the ATP-binding pocket of HCK and blocks ATP binding. (A) Docking model A of the ibrutinib (purple stick) aligned with the co-crystal Thr 333 structure of HCK (green ribbon)-A419259 (yellow stick). Glu 334 The hydrogen bonds (depicted as red dashed lines) 3.1Å between the aminopyrimidine moiety of ibrutinib and the 3.1Å hinge region of HCK are indicated. Docking studies Å showed that ibrutinib bound to the ATP-binding pocket of 3.3 3. A419259 Ibrutinib 2Å Ibrutinib HCK with calculated affinity energy (DG) of 210.5 kcal/mol. Asp 404 (B) Results from pull-down experiments using SVB, IB-BTN, Met336 and CC-292 (CC-BTN) to detect BTK and HCK binding in A419259 MYD88-mutated BCWM.1, MWCL-1, and TMD-8 cells. Asp 348 Asn 391 (C) Results from kinase active-site inhibition assays Ala 390 utilizing an ATP-BTN probe that was used to pull down active kinases in the presence of ibrutinib, CC-292, or A419259 in lysates from BCWM.1 WM cells. ATP-BTN, ATP-biotin; IB-BTN, biotinylated ibrutinib; SVB, streptavidin B BCWM.1 MWCL-1 TMD-8 Lysates beads.

IB-BTN/SVB CC-BTN/SVB IB-BTN/SVB CC-BTN/SVB IB-BTN/SVB CC-BTN/SVB SVB SVB SVB BCWM.1 MWCL-1 TMD-8

BTK

HCK

C +Ibrutinib +CC-292 +A419259 IP: ATP-biotin Compounds (µM) – ––10 1.0 0.1 – 10 1.0 0.1 –– 10 1.0 0.1 ATP-BTN –++++ –++++– ++++

SVB +++ ++ +++ ++ +++++

IB: BTK

input

IB: HCK

input

IB: SRC

input

IB: LYN

input

10,27 calculated affinity energy (DG) of 210.5 kcal/mol. Similar to concentration [IC50] # 0.5 nM). Ibrutinib reversibly inhibits the A419259, ibrutinib’s 4-amino group, forms an hydrogen-bond to kinase activity of HCK with an IC50 of 3.7 nM, whereas CC-292 is a 333 338 the carbonyl groups of the gatekeeper residue Thr (Thr based very weak HCK inhibitor with an IC50 of 14.6 mM, which is well above on c-SRC numbering)15 andalsowithGlu334 of HCK; its 3-nitrogen the observed physiological concentrations of this drug.10,27 As atom, as an hydrogen-bond acceptor, interacts with the backbone expected, biotinylated ibrutinib and CC-292 pulled down BTK in amino group of Met336 of HCK (Figure 6A). The 4-phenoxyphenyl mutated MYD88 expressing BCWM.1, MWCL-1, and TMD-8 cells, substituent at 5-position of ibrutinib, which is also identical to the demonstrating their ability to directly bind to BTK (Figure 6B). 5-substituent of A419259, extends into the inner ATP-hydrophobic Biotinylated ibrutinib, but not CC-292 pulled down HCK in MYD88- pocket of HCK. The pyrrolidine group at the 7-position of ibrutinib, mutated BCWM.1, MWCL-1, and TMD-8 cells, thereby confirming the only distinctive substituent related to A419259, is predicted binding of ibrutinib to HCK (Figure 6B). to interact with Ala390,Asn391,orAsp404 of HCK, whereas the To confirm target engagement in living cells, we performed KiNativ 7-substituent of A419259 protrudes outward the ATP pocket and profiling where the ability of inhibitors to protect kinases from sub- interacts with Asp348 (Figure 6A). sequent labeling with a reactive ATP-biotin probe is determined.28 To confirm HCK target engagement in cells by ibrutinib, we Living cells were treated with either ibrutinib, CC-292, or A419259, synthesized a biotin-modified version of ibrutinib. For comparative followed by lysis treatment with ATP-biotin and western blotting purposes, we also prepared a biotin-modified version of CC-292, a for BTK, HCK, and other SRC family kinases. Consistent with the pyrimidine-based covalent BTK inhibitor.27 Bothibrutinib and CC-292 biochemical kinase assays, ibrutinib and A419259 but not CC-292 use an acrylamide-moeity to form a covalent bond to Cys481 and are blocked ATP binding to HCK in a dose-dependent manner, whereas potent kinase inhibition of BTK (reported half maximal inhibitory ibrutinib and CC-292 but not A419259 blocked ATP binding to BTK in From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3248 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

A DMSO A419259 Ibrutinib CC-292 100 100 MFI: 100 MFI: 100 MFI: 100 MFI: 80 80 2307 80 661 80 1396 80 2460 60 60 60 60 60 40 40 40 40 40 0.089 77.7% 25.1% 65.4% 85.4%

BCWM.1 20 20 20 20 20 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 2675 80 753 80 1214 80 2733 60 60 60 60 60 40 40 40 40 40 0.45 87.1% 35.7% 57.8% 86.9%

MWCL-1 20 20 20 20 20 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 1508 80 750 80 1228 80 1522 60 60 60 60 60 40 40 40 40 40

TMD-8 0 80.7% 9.63% 67.6% 82.1% 20 20 20 20 20 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 1867 80 357 80 917 80 1863 60 60 60 60 60 40 40 40 40 40 0 68.8% 4.04% 48.3% 70.5% OCI-Ly3 20 20 20 20 20 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 1302 80 582 80 873 80 1254 60 60 60 60 60 40 40 40 40 40

HBL-1 0 80.1% 12.2% 48.8% 77.4% 20 20 20 20 20 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105

Control Ab p-Hck DMSO A419259 Ibrutinib CC-292 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 2538 80 526 80 1551 80 2874 60 60 60 60 60 40 40 40 40 40 0.094 96.9% 12.6% 78.9% 97.3% 20 20 20 20 20

WM Patient 1 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 1376 80 828 80 1042 80 1308 60 60 60 60 60 40 40 40 40 40 20 0 20 91.5% 20 54.8% 20 76.7% 20 90.3%

WM Patient 2 0 0 0 0 0 0 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 100 100 100 100 100 MFI: MFI: MFI: MFI: 80 80 1473 80 511 80 1021 80 1378 60 60 60 60 60 40 40 40 40 40 0 86.9% 10.5% 68.8% 83.9% 20 20 20 20 20

WM Patient 3 0 0 0 0 0 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 100 100 100 100 MFI: MFI: MFI: 0.012 MFI: 80 80 632 80 400 80 467 568 0.009 60 60 60 60 40 40 40 40 0.006 0.032 50.6% 15.1% 25.8% 42.2% 0.003 20 20 20 20

WM Patient 4 0 0 0 0 0 3 4 5 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 10 10 10

Control Ab p-Hck

Figure 7. From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3249

HCK Tyr411 phosphorylation in MYD88 L265P expressing WM and B ABC-DLBCL cell lines, as well as primary WM LPC. In contrast, CC-292 Ibrutinib had little or no impact on HCK Tyr411 phosphorylation (Figure 7A). 150 BCWM.1 MWCL-1 Decreased dose-dependent viability was also observed for ibrutinib and TMD-8 more so for A419259 that was more pronounced in mutated MYD88 vs 100 HBL-1 MYD88WT cells (Figure 7B). In contrast, the BTK inhibitor CC-292 OCI-Ly3 OCI-Ly7 that lacks HCK kinase inhibition showed higher half maximal effective OCI-Ly19 . 50 concentration ( 1 log-fold) for the mutated MYD88 cell lines, with the Ramos exception of OCI-Ly3 cells that carry a NF-kB activating CARD11 RPMI-8226 mutation (Figure 7B).5 Increased apoptotic changes were also observed RLUs (DMSO norm) 0 in the mutated MYD88 cell lines and primary WM cells that showed -10 -8 -6 -4 decreased viability following ibrutinib, and A419259 even more so. In Log10 (Dose) (M) contrast, CC-292 had little or no impact on apoptosis in mutated A419259 MYD88 cells (Figure 7C). Also, we observed little or no apoptotic 150 BCWM.1 activity for ibrutinib, A419259, or CC-292 apoptosis in MYD88WT cell MWCL-1 TMD-8 lines or HD B cells (data not shown). To investigate whether ibrutinib or 100 HBL-1 A419259-induced killing of WM cells was a consequence of BTK and/ OCI-Ly3 L265P OCI-Ly7 or HCK inhibition, we transduced MYD88 expressing BCWM.1 OCI-Ly19 WM cells with a lentiviral control vector or vectors expressing WT 50 Ramos BTK; BTK with a mutation of the cysteine required for irreversible RPMI-8226 inhibition (BTKC481S); WT HCK; or HCK with the gatekeeper RLUs (DMSO norm) 0 mutation Thr333 (Thr338 basedonc-SRCnumbering).15 Transduction -10 -8 -6 -4 of BCWM.1 WM cells with the BTK cysteine mutant (BTKC481S)but Log10 (Dose) (M) not the vector control or WT BTK, resulted in decreased ibrutinib and CC-292 CC-292–mediated killing of BCWM.1 WM cells (,1 log-fold shift) 150 BCWM.1 (Figure 7D). In contrast, treatment with HCK inhibitor A419259 showed MWCL-1 TMD-8 no survival change vs vector control transduced cells (Figure 7D). 100 HBL-1 TransductionofBCWM.1WMcells with the HCK gatekeeper OCI-Ly3 T333M T338M 15 OCI-Ly7 mutant (HCK ;HCK basedonc-SRCnumbering ) resulted OCI-Ly19 in a .2 log-fold decrease in both ibrutinib and A419259-mediated 50 Ramos tumor cell killing. Over-expression of WT HCK also resulted in a 1 log- RPMI-8226 fold decrease in both ibrutinib and A419259-mediated tumor cell RLUs (DMSO norm) 0 killing when compared with control vector transduced BCWM.1 WM -10 -8 -6 -4 cells (Figure 7D). No substantial change in tumor cell killing following Log10 (Dose) (M) CC-292 treatment was observed in BCWM.1 cells transduced to express HCKT333M or WT HCK (Figure 7D). Figure 7. (Continued).

BCWM.1 WM cells. Consistent with its known activity, A419259 also blocked ATP binding to the SRC family members SRC and LYN. Conversely, ibrutinib and CC-292 did not block ATP binding to either Discussion SRC or LYN (Figure 6C). We sought to clarify the contribution of non-BTK target(s) to the HCK activity is blocked by ibrutinib and impacts survival of activity of ibrutinib in MYD88-mutated diseases such as WM. We MYD88-mutated WM cells focused this study on HCK, a member of the SRC family of kinases. HCK is over-expressed at early stages of B-cell development, and To assess the impact of ibrutinib and A419259 on HCK-related activity its transcriptional regulation is suppressed with terminal B-cell in MYD88-mutated WM and ABC DLBCL tumor cells, we performed differentiation.29,30 Using gene expression proﬁling, Gutierrez´ et al11 Phosﬂow studies and assessed changes in HCK activation (Tyr411). We previously reported that HCK was highly over-expressed in malignant also evaluated tumor-cell viability by the CellTiter-Glo Luminescent LPC taken from WM patients, although the genetic basis for this Cell Viability Assay, and apoptosis by PI and Annexin V staining observation remained unclear. In this study, we observed that HCK was to determine if HCK inhibition contributed to WM cell death. highly expressed in MYD88-mutated primary WM cells, WM, and We observed that ibrutinib, and A419259 even more so, reduced ABC-DLBCL cell lines, but was absent or expressed at low levels in HD

Figure 7. HCK activity is blocked by ibrutinib and impacts the survival of MYD88-mutated WM cells. (A) Phosflow analysis showing changes in pHCKTyr411 following treatment of MYD88-mutated WM (BCWM.1 and MWCL-1) and ABC DLBCL (TMD-8, OCI-Ly3, and HBL-1) cells and MYD88-mutated primary LPCs from 4 WM patients with 0.5 mM of A419259, ibrutinib, or CC-292 for 30 minutes. (B) Dose-dependent survival determined by CellTiter-Glo Luminescent Cell Viability Assay for mutated (BCWM.1, MWCL-1, TMD-8, HBL-1, and OCI-LY3) and WT (OCI-LY7, OCI-LY19, Ramos, and RPMI 8226) MYD88 cells following treatment with ibrutinib (top), A419259 (middle), or CC-292 (bottom) for 72 hours. (C) Apoptotic changes using PI and Annexin V (FITC-A) staining following treatment of mutated and WT MYD88 cell lines, and primary LPCs from 3 WM patients with 0.5 mM of A419259, ibrutinib, or CC-292 for 18 hours. DMSO denotes vehicle control only treated cells. Cell line results are from experiments performed in triplicate. Primary LPC data are from results obtained from 6 WM patients. *P # .05 and **P # .01 vs DMSO controls. (D) Dose-dependent tumor cell survival of MYD88-mutated BCWM.1 cells transduced with control vector or vectors expressing WT BTK (BTK WT); BTK-expressing mutated site for ibrutinib binding (BTK C481S); WT HCK (HCK WT); or HCK-expressing the gatekeeper mutation (HCKT333M;HCKT338M based on c-SRC numbering)15 and treated with ibrutinib, A419259, or CC-292. Protein levels following transduction with control, WT, or mutated BTK or HCK vectors are also shown at the bottom of (D). FITC-A, fluorescein isothiocyanate A; RLU, relative luminescence units. From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3250 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

C BCWM.1 TMD-8 30 ** 30 ** * 20 20 * 10 10 Primary WM LPC (N = 6) 40 Apoptosis (%) Apoptosis (%) 0 0 ** 30 *

DMSO DMSO A419259 Ibrutinib CC-292 A419259 Ibrutinib CC-292 20

MWCL-1 HBL-1 Apoptosis (%) 10

MYD88 L265P 20 50 ** ** 15 40 0 * 30 ** 10 DMSO 20 A419259 Ibrutinib CC-292 5 10 Apoptosis (%) Apoptosis (%) 0 0

DMSO DMSO A419259 Ibrutinib CC-292 A419259 Ibrutinib CC-292

OCI-Ly19 RAMOS OCI-Ly7 30 30 30

20 20 20 * * 10 ** 10 10 Apoptosis (%) Apoptosis (%) Apoptosis (%) MYD88 WT 0 0 0

DMSO DMSO DMSO Ibrutinib Ibrutinib A419259 CC-292 A419259 CC-292 A419259 Ibrutinib CC-292

D Ibrutinib A419259 150 150 Vector BTK WT Vector 100 100 BTK C481S BTK WT HCK WT BTK C481S HCK WT 50 HCK T333M 50 HCK T333M RLUs (DMSO norm) RLUs (DMSO norm) 0 0 -10 -8 -6 -4 -10 -8 -6 -4 Log10 (Dose) (M) Log10 (Dose) (M)

CC-292 150

Vector 100 BTK WT VectorBTK WTBTK C481S Vector HCK WTHCK T333M BTK C481S HCK WT BTK HCK 50 HCK T333M

RLUs (DMSO norm) GAPDH GAPDH 0 -10 -8 -6 -4 Log10 (Dose) (M)

Figure 7. (Continued). From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25 HCK IS A TARGET OF IBRUTINIB 3251

Bcells,aswellasMYD88WT cell lines. Furthermore, we observed that more so HCK bearing a mutated gatekeeper (HCKT333M;HCKT338M over-expression of the MYD88L265P protein itself markedly induced based on c-SRC numbering)15 reduced the activity of both of these HCK transcription across multiple B-cell lines, including both MYD88- compounds in MYD88-mutated cells. The attenuation of MYD88- mutated and WT cell lines. It is not likely that HCK transcription was directed HCK activity may provide mechanistic insights into the high triggered by MYD88-triggered IL-6, because exogenous treatment of response activity observed in MYD88-mutated WM and ABC- MYD88-mutated WM cells with IL-6 or an IL-6 antibody did not DLBCL patients undergoing ibrutinib therapy.4,9 It also remains directly impact HCK transcription. In addition, CXCR4 mutations that possible that mutations in the gatekeeper site of HCK could contribute are found in up to 40% of WM patients neither impacted HCK to ibrutinib resistance, akin to that observed with BTKC481S mutations transcription or activation in primary WM cells, nor did we observe in chronic lymphocytic leukemia patients, and warrant examination.40 HCK transactivation in previous studies with MYD88-mutated Finally, the potential to develop more potent HCK inhibitors is suggested BCWM.1 and MWCL-1 cells that were transduced with CXCR4 by these findings. Although ibrutinib suppressed HCK activity, higher WHIM-mutated receptors and stimulated with CXCL12 (unpublished levels of HCK suppression were observed with A419259, and were observations).31,32 Our findings therefore provide a genomic explana- associated with greater levels of apoptosis in MYD88-mutated WM tion for HCK over-expression in WM, and suggest that mutated MYD88 and ABC-DLBCL cells. Several scaffolds for targeted HCK inhibition enforces high transcription levels of HCK. NF-kB, activator protein 1, are in development based on prior work implicating HCK in the and cAMP-response element binding sites are present on the promoter of pathogenesis of several solid and hematologic malignancies, as well as HCK, and their transactivation isknowntobetriggeredbyWTMYD88 productive HIV infection.41,42 In addition to inhibiting other SRC in response to toll-like receptor stimulation.31-35 Studies to delineate the family members, dasatinib also shows potent inhibition of HCK and precise signaling cascade(s) by which mutated MYD88 transduces may be active in diseases dependent on mutated MYD88.43 HCK are therefore needed, although the constitutive activation of In conclusion, our findings suggest that HCK is a downstream target NF-kB by mutated MYD88 is a strong suspect in WM and ABC DLBCL of mutated MYD88, is activated by IL-6, and triggers pro-survival cells, as well as activator protein 1 in ABC DLBCL.1,5,36 The BCR and signaling including PI3K/AKT, MAPK/ERK, and BTK in MYD88- other pro-survival pathways may also contribute to HCK activation mutated cells. HCK is also a highly relevant target of ibrutinib, and and warrant further investigation. represents a novel focus for therapeutic development in WM and The findings also suggest an important role for IL-6 in triggering possibly other diseases driven by mutated MYD88. HCKactivityinWM,andpossiblyother MYD88-mutated diseases. We show that bothIL-6 and IL-6R are highlyexpressed inWM disease, and that MYD88 L265P itself induces IL-6 transcription. Ngo et al5 showed that HCK and IL-6 were among 285 genes whose transcription was downregulated upon knockdown of MYD88 in HBL-1 ABC- Acknowledgments DLBCL cells that express L265P as a heterozygous mutation. The role of IL-6 as an activator of pHCK through IL-6R/IL-6ST has previously The authors gratefully acknowledge the generous support of Peter Bing, the been reported, and appears dependent on an acidic domain (amino International Waldenstrom¨ ’s Macroglobulinemia Foundation, the Leuke- acids 771-811) of IL-6ST.12,13 IL-6 is highly transcribed in WM and mia and Lymphoma Society, the Edward and Linda Nelson Fund for WM ABC-DLBCL tumors.11,37,38 IL-6 is also secreted by the BM Research, the Bauman Family Trust, the Tannenhauser Family Founda- microenvironment in response to WM cell adhesion.14 We show in tion, and the WM patients who provided samples for these studies. The these studies that HCK is hyper-activated in mutated MYD88 primary authors also acknowledge the kind gift of Thomas Smithgall for providing WM cells as well as WM and ABC-DLBCL cell lines, and that the HCK inhibitor-resistant mutant construct used in these studies. exogenous IL-6 induced its activation, whereas knockdown of IL-6ST attenuated HCK activation. HCK was also found to induce PI3K/AKT, MAPK/ERK, and BTK signaling, providing for the first time evidence that mutated MYD88 can trigger pro-survival signaling beyond its known activation of NF-kB by eliciting HCK transcription and Authorship activation. The transactivation of BTK by HCK is also noteworthy. Cheng et al previously described that BTK could bind to HCK through Contribution: G.Y., S.J.B., L.T., N.G., and S.P.T. conceived and SH3-mediated interactions.21 In previous work, we described that the designed the experiments; G.Y. and S.P.T. wrote the manuscript; activated-form BTK was complexed with MYD88 in MYD88- G.Y., Z.R.H., and S.P.T. performed the data analysis; X.L. and N.T. mutated cells, although the mechanism for BTK activation in this performed PCR-based sequencing studies; G.Y., X.L., and J.C. context remained unclear. Our findings offer a mechanistic explanation performed transduction experiments; X.L. and J.G.C. performed for BTK activation in MYD88-mutated WM and ABC DLCBL cells. western blot studies; G.Y. and J.C. performed Phosflow studies; Akeyfinding of this study was the recognition of HCK as a novel L.T., S.J.B., and N.G. performed docking studies; L.X., X.L., and therapeutic target for WM, and possibly other mutated MYD88 diseases. C.J.P. prepared samples; S.P.T., J.R.B., and J.J.C. provided patient Kinase selectivity profiling had previously implicated HCK as a potential care, and obtained consent and samples; W.Z., X.Z., and S.L. target of ibrutinib with an IC50 of 3.7 nM, a dose level close to the IC50 of provided biotinylated ibrutinib; and P.C. provided BTK-C481S BTK (0.5 nM) and well within the pharmacologically achievable levels and HCK-T333M vectors for transduction experiments. of ibrutinib.10,39 We provide definitive evidence in this report that Conflict-of-interest disclosure: S.P.T. and J.R.B. have received HCK is a target of ibrutinib using docking models, direct pull-down research funding, consulting fees, and/or honoraria from Pharmacy- experiments with biotinylated ibrutinib, cellular target engage- clics Inc., and Janssen Oncology Inc. The remaining authors declare ment, and cellular rescue with inhibitor-resistant mutant proteins. no competing financial interests. We also observed that treatment of mutated MYD88 WM and ABC- Correspondence: Steven P. Treon, Dana Farber Cancer Institute DLBCL cells with ibrutinib, or a preclinical HCK inhibitor (A419259) and Harvard Medical School, 450 Brookline Ave, M546, Boston, attenuated HCK activation, whereas over-expression of WT HCK and MA 02215; e-mail: [email protected]. From www.bloodjournal.org by Jorge Castillo on June 24, 2016. For personal use only.

3252 YANG et al BLOOD, 23 JUNE 2016 x VOLUME 127, NUMBER 25

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2016 127: 3237-3252 doi:10.1182/blood-2016-01-695098 originally published online May 3, 2016

HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib

Guang Yang, Sara J. Buhrlage, Li Tan, Xia Liu, Jie Chen, Lian Xu, Nicholas Tsakmaklis, Jiaji G. Chen, Christopher J. Patterson, Jennifer R. Brown, Jorge J. Castillo, Wei Zhang, Xiaofeng Zhang, Shuai Liu, Philip Cohen, Zachary R. Hunter, Nathanael Gray and Steven P. Treon

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