The interplay between microenvironmental signaling and novel targeted drugs in CLL - Thesis (complete)

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The interplay between microenvironmental signaling and novel targeted drugs in

CLL

Thijssen, R.

Publication date

2016

Document Version

Final published version

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Citation for published version (APA):

Thijssen, R. (2016). The interplay between microenvironmental signaling and novel targeted

drugs in CLL.

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The inTerplay beTween

microenvironmenTal

signaling and novel

TargeTed drugs in cll

rachel Thijssen

R

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The in

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The inTerplay beTween

microenvironmenTal

signaling and novel

TargeTed drugs in cll

Lay out and printing: Off page, Amsterdam Cover design: Bert Potse

ISBN: 978-94-6182-733-3

The printing of this thesis was financially supported by: AbbVie, Celgene, Gilead Sciences, MRC-Holland, Roche, Sanofi-Genzyme

Copyright © Rachel Thijssen, Amsterdam, The Netherlands

All right reserved. No part of this thesis may be reproduced or transmitted in any from or by any means without the written permission of the author.

THE INTERPLAY BETWEEN MICROENVIRONMENTAL SIGNALING

AND NOVEL TARGETED DRUGS IN CLL

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel

op vrijdag 25 november 2016, te 14:00 uur

door

Rachel Thijssen

PROMOTIECOMMISSIE

Promotores

Prof. dr. E.F. Eldering Universiteit van Amsterdam Prof. dr. A.P. Kater Universiteit van Amsterdam

Copromotor

Prof. dr. M.J. Kersten Universiteit van Amsterdam

Overige leden

Prof. dr. M.H.J. van Oers Universiteit van Amsterdam Prof. dr. J. Borst Universiteit van Amsterdam Dr. M. Spaargaren Universiteit van Amsterdam Prof. dr. J.H. Veelken Universiteit Leiden

Prof. dr. R.W. Hendriks Erasmus Universiteit Rotterdam Faculteit der Geneeskunde

TABLE OF CONTENTS

Chapter 1 General Introduction 7

Chapter 2 The pan phosphoinositide 3-kinase/mammalian 21

target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells

Chapter 3 Dual TORK/DNA-PK inhibition blocks critical signalling 51 pathways in chronic lymphocytic leukemia

Chapter 4 Targeting antigen-independent proliferation in chronic 83 lymphocytic leukemia through differential kinase inhibition

Chapter 5 Resistance to ABT-199 induced by microenvironmental signals 103 in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors

Chapter 6 IL-21 and IL-4 distinctly affect NF-

κ

B binding on the Bcl-XL 121 promoter in CLL cells

Chapter 7 General Discussion 139

Appendices Summary 157 Samenvatting 161 Curriculum Vitae 164 Publications 165 PhD Portfolio 166 Dankwoord 168

GENERAL INTRODUCTION

1

GENERAL INTRODUCTION

1

INTRODUCTION

Chronic lymphocytic leukemia (CLL), the most common adult leukemia in western countries, is characterized by the accumulation of mature, CD5+_{, CD23}+ _monoclonal

B lymphocytes in the blood, secondary lymphoid tissues, and bone marrow1_{. There is}

considerable heterogeneity in the clinical course and patients’ response to therapy. Three patient groups can be distinguished based on age, comorbidities and life expectancy regardless of the diagnosis of cancer2,3_{. The first group are patients <65 years of age}

and physically fit without or with mild comorbidities. First line treatment for this group is chemoimmunotherapy, with fludarabine, cyclophosphamide, and rituximab (FCR) 2,3_.

The second group are treatment-naïve fit patients older than 65 years and first line treatment is the combination of bendamustine and rituximab (BR)2-4_{. The third group are}

patients with multiple or severe comorbidities and this group receives chlorambucil with a CD20 antibody as first line treatment2,3_{. FCR is considered the best treatment option}

with a long progression free survival and 1/3 of patients that might experience long lasting disease control. However, CLL remains an incurable disease and relapses are common after FCR and the alternative regimens5_{. Eventually, CLL cells develop drug resistance}

which results in a very poor prognosis6_{. At least two mechanisms are believed to contribute}

to the development of resistance to drugs. First, patients carrying specific chromosomal abnormalities respond poorly to chemoimmunotherapy. Such chromosomal abnormalities are deletion of 17p or 11q, which contain the genes for the tumor suppressor p53 and the DNA damage sensing kinase ATM, respectively7,8_{. Second, development of drug}

resistance in CLL is also strongly dependent on the lymphoid microenvironment.

Key signal transduction pathways in CLL

In CLL, two compartments can be distinguished: the blood, in which quiescent CLL cells accumulate, and the lymphoid microenvironment within the lymph nodes (LN) and spleen where proliferation occurs. In the LN, CLL cells interact with non-leukemic accessory cells, such as stromal cells9_{, monocyte-derived nurse-like cells}10_{, and CD40L expressing T cells}11

and provide key survival signals to the CLL cells12-14 _{(Figure 1). Stimulation of the B cell}

receptor (BCR), tumor necrosis factor (TNF) family members or cytokine receptors induce multiple downstream signaling cascades that drive survival and proliferation of CLL cells15_.

These signaling cascades lead to proliferation, adhesion and a shift in the apoptotic balance with upregulation of pro-survival proteins14,16_{. Here, we discuss key signal transduction}

pathways in CLL that drive adhesion, proliferation and survival.

BCR signaling

The BCR is a multimeric complex composed of a surface immunoglobulin and the BCR subunit CD79. Upon binding of an antigen to the immunoglobulin, LYN, a kinase from the BCR complex, phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tail of CD79, which then recruits Spleen tyrosine kinase (SYK) (Figure 2)17_{. As a next step in this pathway, LYN and SYK transduce the signal leading to}

activation of extracellular signal-regulated kinases (ERK) or Bruton’s tyrosine kinase (BTK)18_.

GENERAL INTRODUCTION

1

(BLNK) adaptor, resulting in downstream effector responses, including calcium signaling (Ca2+_{), integrin and Protein kinase C (PKC) activation}19,20_{. The BCR-associated co-receptor}

CD19 contributes to the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway21,22_{. Mamalian target of rapamycin complex (mTORC) 1 (Raptor) is activated by}

AKT, which leads to phosphorylation of downstream effectors, such as 4EBP1 and S623_.

In addition, mTORC2 (Rictor) activation leads to phosphorylation of AKT on the serine

site24,25_{. The PI3K/AKT/mTOR pathway is important for cellular survival, metabolism and}

proliferation in normal B-cells21_{. Activation of the BCR occurs in the microenvironment,}

where CLL cells encounter auto-antigens or environmental antigens16,26,27_{. In addition to}

antigen-dependent signaling, autonomous BCR signaling due to BCR crosslinking with BCR-intrinsic motifs has been reported28_.

CD40 signaling

In the lymph node, CLL cells are in close contact with activated CD4+ _{T cells that}

express the CD40 ligand (CD40L) on their surface11_{. The CLL cells themselves produce}

the chemokines C-C chemokine ligand (CCL) 4 and CCL22 to attract the CD4+ _{T cells}

and induce the CD40L-CD40 interaction11_{. CD40 is a member of the tumor necrosis factor}

receptor (TNFR) family and is expressed on the membrane of CLL cells29_{. Upon binding}

Figure 1. Schematic representation of the CLL lymph node microenvironment. Two compartments can be distinguished: the blood, where the cells are in rest phase, and the lymphoid microenvironment. In the lymph node microenvironment, survival of CLL cells is induced through B cell receptor (BCR) triggering or signals provided by surrounding cells. T cells interact with CLL cells through CD40L-CD40 binding or by production of the cytokines IL-21/IL-4. Nurse like cells induce survival via BAFF or APRIL or the production of chemokines CXCL12/CXCL13. Homing of CLL cells to the lymph node is mediated by the chemokine production of stromal cells and the binding of integrin to VCAM1/ ICAM1. These signaling cascades lead to the upregulation of the anti-apoptotic regulators Bcl-XL, Bfl-1 and Mcl-1, resulting in survival of the CLL cell.

GENERAL INTRODUCTION

1

of CD40L, the CD40 molecule trimerizes on lymphocytes and recruits the adaptor protein tumor necrosis factor receptor-associated factors (TRAFs) to the cytoplasmic domain of CD4030_{. TRAFs activate different signaling pathways including the mitogen-activated}

protein kinase (MAPK) signaling, the PI3K/AKT pathway, and the Nuclear factor-kappa B (NF-

κ

B) signaling pathway30_{. TRAF1, 2 and 6 are important for the canonical NF-}

κ

_B

signaling pathway and activate the transforming growth factor-

β

-activated kinase 1 (TAK1) which leads to subsequent I

κ

B kinase (IKK) phosphorylation (Figure 3)31,32_{. Activated IKK,}

consisting of IKK

α

, IKK

β

, and IKK

γ

, phosphorylates I

κ

B which is part of a complex with NF-

κ

B1 p50 and p65. Upon phosphorylation, I

κ

B is degraded leading to the translocation of the p50/p65 dimer to the nucleus where activation of target genes occurs, including the anti-apoptotic regulator Bfl-131-33_.

TRAF2 and TRAF3 are important modulators of the non-canonical NF-

κ

B pathway. Upon CD40 activation, TRAF2, TRAF3, and cIAP1/2 are recruited to the receptor, where cIAP1/2-mediated degradation of TRAF2 and TRAF3 occurs34_{. Without CD40 activation,}

NIK is degraded through the TRAF2/TRAF3/cIAP1/2 complex. Upon CD40 activation, NIK stabilizes and induces phosphorylation of the IKK

α

homodimers and subsequent p100 phosphorylation35_{. Upon phosphorylation, p100 is transformed in p52 and together with}

RelB will translocate to the nucleus where the p52-RelB complex activates target genes, including the anti-apoptotic regulator Bcl-XL (Figure 3)33,35,36_.

In vitro CD40L stimulation is used to mimic the microenvironment and shifts the balance

to a more anti-apoptotic profile in PB CLL cells as seen in LN CLL cells 14,37-39_{. CD40}

triggering causes upregulation of the anti-apoptotic proteins Mcl-1, Bfl-1, and Bcl-XL and downregulation of the pro-apoptotic protein Noxa. These changes result in increased drug resistance40,41_.

Toll-like receptor 9 signaling

The innate Toll-like receptor (TLR) 9 is activated through unmethylated cytosine guanine dinucleotide (CpG) motifs in bacterial DNA and oligonucleotides. CpG-rich hypomethylated DNA motifs are a potent activator of CLL cells42_{. Upon TLR9 activation,}

Figure 2. Schematic representation of the B cell receptor pathway. Upon B cell receptor (BCR) and CD79 activation, LYN and SYK are recruited and phosphorylated. Activated LYN/SYK phosphorylate ERK or BTK. Downstream of BTK, PLC

γ

2 is activated via BLNK. CD19 activates the PI3K/AKT/mTOR pathway.

GENERAL INTRODUCTION

1

the Toll–IL-1-resistence (TIR) domain of TLR9 engages the TIR domain-containing adaptor protein myeloid differentiation marker 88 (MyD88)43_{. MyD88 contains an IL-1R-associated}

kinase 1 (IRAK1) domain which interacts with TRAF6. Upon activation of TRAF6, the NF-

κ

B, p38 MAPK and JUN N-terminal kinase (JNK) pathways are activated43_{. CpG stimulation}

in unmutated IgVH CLL cells results in proliferation, while CpG stimulation in mutated IgVH CLL cells results in apoptosis39_{. An explanation for this is that the}

ζ

_{-chain-associated}

protein (ZAP) 70 is aberrantly expressed in unmutated CLL cells and important for the TLR9-mediated activation of SYK44_{. Very recently, it was shown that SYK activation leads to}

production and secretion of autoreactive IgM and this provides an anti-apoptotic signal44_.

Cytokine receptor signaling

In the microenvironment, CLL cells are in close contact with CD4+ _{T cells that express}

high levels of CXCR545_{. It is assumed that these T cells are T follicular helper cells (Tfh)}

which produce cytokines that are important for the T-B cell interaction46_{. One major}

cytokine produced by Tfh cells is the class I cytokine interleukin 21 (IL-21). CLL cells upregulate the IL-21 receptor (IL-21R) after CD40L stimulation47_{. Signaling via the IL-21}

receptor complex involves activation of JAK1 and JAK3 and results in phosphorylation of STAT-1, STAT-3, and STAT-547_{. Our studies have shown that IL-21-mediated signaling}

can be found in LN samples isolated from CLL patients48_{. In addition, CD40L and IL-21}

can initiate proliferation of CLL cells in vitro48_{. Another important cytokine involved in}

Figure 3. Schematic representation of CD40 signaling. Upon CD40 receptor stimulation, TRAF is translocated to the receptor and can activate the canonical or non-canonical NF-

κ

B pathway. Activation of the canonical pathway leads to degradation of IKK

γ

and phosphorylation of IKK

β

. This in turns phosphorylates and degrades I

κ

B what leads to translocation of p50/p65 to the nucleus and can activate their target genes. Upon activation of the non-canonical NF-

κ

B pathway, NIK is stabilized and IKK

α

is phosphorylated. P100 is transformed in p52 and p52 together with RelB translocate to the nucleus and can activate their target genes.

GENERAL INTRODUCTION

1

the T-B cell interaction is IL-4 produced by T cells49_{. IL-4 receptor (IL-4R) stimulation}

results in JAK1 and JAK 3-mediated phosphorylation of STAT1, STAT5, and STAT650_{. CLL}

cells show increased expression of IL-4R compared with healthy B cells which correlates with increased pSTAT6 expression49,51_{. In vitro IL-4 stimulation results in increased cellular}

survival52_{. In CLL patients, an increased number of IL-4-producing T cells in the blood is}

a marker of poor prognosis53_.

APOPTOSIS REGULATION

In the CLL microenvironment, activation of key signal transduction pathways changes the balance of apoptotic regulators. The balance of apoptotic regulators in a cell is a key determinant of survival and drug resistance. Apoptosis occurs via either triggering of cell surface death receptors (i.e., the extrinsic pathway) or perturbation of mitochondria followed by cytochrome C release and caspase activation (i.e., the intrinsic pathway)54_{. Upon}

induction of the intrinsic apoptosis pathway, Bax and Bak form pores in the mitochondria through which cytochrome C can be released54,55_{. Some pro-apoptotic BH3-only proteins}

can indirectly initiate apoptosis by binding to anti-apoptotic Bcl-2 proteins. These interactions prevent the anti-apoptotic proteins from inhibiting the pro-apoptotic Bax and Bak (Figure 4)55_{. Other pro-apoptotic proteins can directly induce apoptosis by binding}

to Bax and Bak56,57_{. Most CLL patients show overexpression of the anti-apoptotic protein}

Bcl-2, due to deleted or silenced miR-15a and/or miR-16.1, which normally suppress Bcl-2 expression58_{. A prominent strategy in CLL treatment is direct targeting of Bcl-2 by use}

of so-called BH3-mimetics. The first generation of BH3 mimetics included ABT-737/263 or navitoclax, which efficiently antagonizes Bcl-2, Bcl-XL, and Bcl-W. The clinical results were encouraging, also in terms of responsiveness in chemorefractory CLL59_{. However,}

platelets also express Bcl-XL which resulted in rapid platelet death. This resulted in dose-limiting thrombocytopenia and the restriction of ABT-737 use in patients with CLL60_.

A second generation BH3-mimetic was developed which lacked binding to Bcl-XL61_{. This}

Bcl-2-directed compound ABT-199/venetoclax is highly cytotoxic for CLL cells and shows improved clinical efficacy61_.

NOVEL KINASE INHIBITORS

Targeting kinases in the key signaling pathway in CLL cells, especially in the BCR pathway, have emerged as promising treatment options. One of the drugs that emerge as a promising treatment option is ibrutinib, a selective and irreversible inhibitor of BTK62_{. Ibrutinib has}

been approved by the FDA for treatment of patients with CLL associated with a poor prognosis – including those with relapsed/refractory CLL and treatment-naive CLL with a deletion 17p or TP53 mutation63_{. Recently, it was shown that ibrutinib induces a longer}

progression free survival in treatment-naïve older patients compared to chlorambucil64_.

These data suggest ibrutinib as a promising therapeutic option for these patients with relapsed/refractory CLL.

Clinical activity of ibrutinib in CLL is attributed to attenuated retention and homing of cells to the CLL microenvironment due to impaired BCR-controlled integrin-mediated adhesion to fibronectin and CD49d/vascular cell adhesion molecule 1 (VCAM-1)65_{. Fibronectin and}

GENERAL INTRODUCTION

1

VCAM-1 both play an important role in the homing of CLL cells to the microenvironment 66_.

Ibrutinib also inhibits chemokine-controlled migration65_{. The inhibitory effects of ibrutinib}

on adhesion and tissue homing correlates with the clinical efficacy. Ibrutinib treatment causes a rapid reduction in the lymph node size followed by a prolonged lymphocytosis62,67_.

Idelalisib is another kinase inhibitor that has emerged as a promising treatment option. This FDA-approved drug targets PI3K

δ

, a kinase in the BCR pathway. Similar to ibrutinib, idelalisib abolishes both chemotaxis towards stroma and BCR-controlled integrin-mediated cell adhesion. This causes a rapid egress of leukemic cells from their protective microenvironment.

The prolonged lymphocytosis as the result from kinase-inhibitor treatment appears to have no clinical disadvantage68,69_{. However, prolonged lymphocytosis could enhance}

the chance of patients accumulating resistant clones. Acquired resistance to ibrutinib was reported in patients with a C481S mutation in the binding pocket of BTK or activating mutations in kinases downstream of BTK70_{. However, in patients that acquired resistance to}

idelalisib, no mutations were observed in the CLL cells. Resistance to idelalisib may occur via a compensatory activation of other PI3K isoforms. Because ibrutinib and idelalisib have limited direct cytotoxic effects on CLL cells, continuous treatment is needed to control the residual disease until patients relapse68-70_{. The long-term clinical application}

of ibrutinib and idelalisib can result in toxicities that can affect the quality of life. Chronic diarrhea has been reported as a severe toxicity for idelalisib treatment and for patients on ibrutinib treatment fatigue was the major toxicity reported69,71,72_{. Because of these}

toxicities, patients have to stop treatment, which can result in the outgrowth of clones that are resistant to ibrutinib or idelalisib. Another issue related to continuous treatment with ibrutinib or idelalisib is the high costs that can burden the healthcare systems. Therefore, there is still a major clinical need for development of novel more effective targeted and/ or combination therapies that result in deep remission and allow for discontinuation with the drugs.

Figure 4. Schematic representation of the apoptotic pathway. Pro-apoptotic BH3-only members Bim, tBid, Puma, Bad, Noxa differentially antagonize anti-apoptotic proteins and either directly or indirectly induce oligomerization of Bax and Bak, leading to apoptosis. Bcl-2, Bcl-XL, Bcl-W, A1, Mcl-1 directly sequester Bax and Bak, or inhibit the pro-apoptotic BH3 domain-only proteins.

GENERAL INTRODUCTION

1

Various companies are currently developing novel inhibitors, which act either upstream (such as SYK-inhibitors) or more downstream (such as mTOR inhibitors) of the BCR and TNF-receptor family pathway15_{. For all these novel emerging therapies, it is of utmost}

importance to understand the signaling pathways in CLL leading to clinical-relevant biological features in these cells, in order to understand how we can avoid the development of drug resistance, for example through combination of agents with different modes of action.

OUTLINE OF THIS THESIS

There is great interest in novel therapies and significant progress has been made in the development of targeted therapy options in CLL, in particular through the development of kinase inhibitors and BH3 mimetics. However, none of these therapies do fully eradicate the disease and resistance has been reported. In this thesis, we studied mechanisms that drive drug resistance and we investigated combination therapy of inhibitors that targeted different signaling routes in the CLL cells. By combination therapy, we hope to overcome resistance mechanisms from coming into play and eventually lead to long-term disease control and discontinuation with drugs.

In chapter 2, a pan-PI3K inhibitor was compared with the more selective PI3K

α

and PI3K

δ

inhibitors, on the impact of induction of apoptosis, and inhibition of cell adhesion, CD40-induced survival and proliferation in primary patient derived CLL cells. In chapter 3, we evaluated the applicability of a novel dual TORK and DNA-PK inhibitor in primary CLL samples of different prognostic risk subgroups. The potency of a dual TORK and DNA-PK inhibitor was analysed with respect to induction of cytotoxicity, and blocking of CD40-mediated chemo-resistance and proliferation. Furthermore, clinical efficacy of DNA-PK/mTOR inhibitor was tested in a clinical trial with CLL patients. As most kinase inhibitors exert an effect on the proliferation of CLL cells, we studied the mechanism underlying antigen-independent proliferation in chapter 4. We studied two distinct antigen-independent proliferation routes in CLL: CD40 + JAK/STAT pathway and the CpG + BCR pathway.

CLL cells are highly sensitive to the Bcl-2-selective BH3-mimetic ABT-199; however, in the microenvironment , pro-survival signals can upregulate Bcl-2 members. In chapter 5 we analyzed if CD40-stimulated CLL cells can become resistant to ABT-199. In chapter 6 we studied the regulation of Bcl-XL; the main regulator of drug resistance in CLL.

GENERAL INTRODUCTION

1

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40. Tromp JM, Geest CR, Breij EC, et al. Tipping the Noxa/Mcl-1 balance overcomes ABT-737 resistance in chronic lymphocytic leukemia. Clin Cancer Res. 2012;18(2):487-498.

41. Hallaert DY, Jaspers A, van Noesel CJ, van Oers MH, Kater AP, Eldering E. c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches. Blood. 2008;112(13):5141-5149.

42. Decker T, Schneller F, Sparwasser T, et al. Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells. Blood. 2000;95(3):999-1006.

43. O’Neill LAJ, Golenbock D, Bowie AG. The history of Toll-like receptors [mdash] redefining innate immunity. Nat Rev Immunol. 2013;13(6):453-460.

44. Wagner M, Oelsner M, Moore A, et al. Integration of innate into adaptive immune responses in ZAP-70-positive chronic lymphocytic leukemia. Blood. 2016;127(4):436-448.

45. Ahearne MJ, Willimott S, Piñon L, et al. Enhancement of CD154/IL4 proliferation by the T follicular helper (Tfh) cytokine, IL21 and increased numbers of circulating cells resembling Tfh cells in chronic lymphocytic leukaemia. British Journal of Haematology. 2013;162(3):360-370. 46. Jin H, Carrio R, Yu A, Malek TR. Distinct activation signals determine whether IL-21 induces B

cell costimulation, growth arrest, or Bim-dependent apoptosis. J Immunol. 2004;173(1):657-665. 47. de Totero D, Meazza R, Zupo S, et al. Interleukin-21 receptor (IL-21R) is up-regulated by

CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells. Blood. 2006;107(9):3708-3715.

48. Pascutti MF, Jak M, Tromp JM, et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood. 2013;122(17):3010-3019.

49. Douglas RS, Capocasale RJ, Lamb RJ, Nowell PC, Moore JS. Chronic lymphocytic leukemia B cells are resistant to the apoptotic effects of transforming growth factor-beta. Blood. 1997;89(3):941-947. 50. Kay NE, Pittner BT. IL-4 biology: impact on normal and leukemic CLL B cells. Leuk

Lymphoma. 2003;44(6):897-903.

51. Bhattacharya N, Reichenzeller M, Caudron-Herger M, et al. Loss of cooperativity of secreted CD40L and increased dose-response to IL4 on CLL cell viability correlates with enhanced activation of NF-kB and STAT6. Int J Cancer. 2015;136(1):65-73.

52. Steele AJ, Prentice AG, Cwynarski K, et al. The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment. Blood. 2010;116(22):4569-4577.

53. Rossmann ED, Lewin N, Jeddi-Tehrani M, Osterborg A, Mellstedt H. Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B-CLL). Eur J Haematol. 2002;68(5):299-306. 54. Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;116(2):205-219.

55. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9(1):47-59.

GENERAL INTRODUCTION

1

57. Green DR. At the gates of death. Cancer Cell. 2006;9(5):328-330.

58. Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A. 2005;102(39):13944-13949.

59. Roberts AW, Seymour JF, Brown JR, et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J Clin Oncol. 2012;30(5):488-496.

60. Schoenwaelder SM, Jarman KE, Gardiner EE, et al. Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets. Blood. 2011;118(6):1663-1674.

61. Roberts AW, Davids MS, Pagel JM, et al. Targeting BCL2 with Venetoclax in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med. 2016;374(4):311-322.

62. Burger JA. Bruton’s tyrosine kinase (BTK) inhibitors in clinical trials. Curr Hematol Malig Rep. 2014;9(1):44-49.

63. Spaargaren M, de Rooij MF, Kater AP, Eldering E. BTK inhibitors in chronic lymphocytic leukemia: a glimpse to the future. Oncogene. 2015;34(19):2426-2436.

64. Burger JA, Tedeschi A, Barr PM, et al. Ibrutinib as Initial Therapy for Patients with Chronic Lymphocytic Leukemia. N Engl J Med. 2015;373(25):2425-2437.

65. de Rooij MF, Kuil A, Geest CR, et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood. 2012;119(11):2590-2594.

66. Vincent AM, Cawley JC, Burthem J. Integrin function in chronic lymphocytic leukemia. Blood. 1996;87(11):4780-4788.

67. Hoellenriegel J, Meadows SA, Sivina M, et al. The phosphoinositide 3’-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood. 2011;118(13):3603-3612.

68. Herman SE, Niemann CU, Farooqui M, et al. Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study. Leukemia. 2014;28(11):2188-2196.

69. Furman RR, Sharman JP, Coutre SE, et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N Engl J Med. 2014;370(11):997-1007.

70. Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton’s tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370(24):2286-2294.

71. Brown JR, Byrd JC, Coutre SE, et al. Idelalisib, an inhibitor of phosphatidylinositol 3-kinase p110delta, for relapsed/refractory chronic lymphocytic leukemia. Blood. 2014;123(22):3390-3397. 72. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic

THE PAN PHOSPHOINOSITIDE 3-KINASE/

MAMMALIAN TARGET OF RAPAMYCIN

INHIBITOR SAR245409 (VOXTALISIB/XL765)

BLOCKS SURVIVAL, ADHESION

AND PROLIFERATION OF PRIMARY CHRONIC

LYMPHOCYTIC LEUKEMIA CELLS

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

R Thijssen1,3,9_{, J ter Burg}1,3,9_{,GGW van Bochove}1,3_{, MFM de Rooij}2_{, A Kuil}2_{, MH Jansen}1_,

TW Kuijpers4,5_{, JW Baars}6_{, A Virone-Oddos}7_{, M Spaargaren}2,9_{,C Egile}8_{, MHJ van Oers}3,9_{, E}

Eldering1,9_{, MJ Kersten}3,9*_{, AP Kater}3,9*

Departments of Experimental Immunology1_{, Pathology}2 _{and Hematology}3_{, Academic Medical Center,}

University of Amsterdam, Amsterdam, The Netherlands.

4 _{Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Amsterdam,}

The Netherlands.

5 _{Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children’s Hospital,}

Academic Medical Center, Amsterdam, The Netherlands.

6 _{Department of Medical Oncology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital,}

Amsterdam, the Netherlands.

Sanofi Oncology, 7 _{Cancer Biology and} 8 _{Translational Medicine, Vitry/Seine, France.} 9 _{Lymphoma and Myeloma Center Amsterdam, LYMMCARE, The Netherlands.} * _{These authors share last authorship on the paper.}

Leukemia 2016, 30(2):337-45

ABSTRACT

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and play an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3K

δ

block BCR-mediated cross-talk between CLL cells and the lymph node (LN) microenvironment and provide significant clinical benefit to CLL patients. However, the PI3K

δ

inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here, we demonstrate that the pan-PI3K/mTOR inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells - irrespective of their ATM/p53 status - than PI3K

α

or PI3K

δ

isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

INTRODUCTION

Chronic lymphocytic leukemia (CLL), the most common adult leukemia in western countries, remains an incurable disease1_{. For their survival and proliferation, CLL cells}

highly depend on both B cell receptor (BCR) mediated signaling as well as on stimuli from the tumor microenvironment within the lymph nodes (LN), spleen and bone marrow. Within this microenvironment, supporting cells such as stromal cells2_{, monocyte-derived}

nurse-like cells3_{, and CD40L expressing T cells}4 _{delivercritical signals protecting CLL cells}

from apoptosis and cell cycle arrest.

New agents targeting key signaling kinases, such as ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK), and idelalisib, an inhibitor of phosphatidylinositol 3-kinase

δ

(PI3K

δ

), have emerged as promising treatment options. The PI3K pathway plays a key role in essential cellular functions including cell growth, migration and survival5_{. Class I PI3Ks are responsible}

for the production of phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P₂), and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) in response to external cell stimuli. In mammalian cells, four class I PI3K isoforms exist, of which PI3K

α

and PI3K

β

are ubiquitously expressed, while PI3K

γ

and PI3K

δ

expression is restricted to hematopoietic cells5,6_{. Although PI3K}

γ

_{is not essential for signaling via}

antigen and cytokine receptors in B cells, it is a key messenger in BCR signaling7,8_{. The PI3K}

pathway is activated in lymphoma and CLL patients9,10 _{due to constitutive BCR activation,}

CD40 ligation11,12 _{and integrin and chemokine receptor activation}13_.

Inhibition of PI3K

δ

by idelalisib abolishes both chemotaxis towards stroma and BCR-controlled integrin-mediated cell adhesion; this prevents cross-talk between CLL cells and protective stromal cells, thereby abrogating pro-survival signaling, and results in a rapid egress of leukemic cells from their protective microenvironment 13-15_{. Despite its significant}

clinical activity, idelalisib has limited direct cytotoxic effects on CLL cells9,13,14 _{which could}

potentially result in the emergence of resistant clones. Acquired resistance to ibrutinib was recently reported in patients due to a C481S mutation in the binding pocket of BTK or due to activating mutations in kinases downstream of BTK16_{. CLL cells with dysfunctional ATM}

or p53 seem to be more prone to developing resistance towards these drugs, probably due to increased genomic instability16_{. Besides specific mutations, acquired resistance to}

PI3K

δ

inhibitors might also arise via a compensatory activation of other PI3K isoforms, as was recently reported in breast cancer with the PI3K

α

inhibitor BYL71917_{. In solid tumors,}

both PI3K

α

and PI3K

β

provide prosurvival signals18_{. In B-cell malignancies, the relative}

importance of PI3K

α

,

β

, and

γ

isoforms is not clear. PI3K

α

is functionally important for B cell development as combined deletion of genes encoding PI3K

α

and PI3K

δ

results in a near complete block of the B cell development in mice, whereas single gene deletion only has a minimal effect19_{. Molecular alterations in components of the PI3K pathway}

are rare in CLL and B-cell lymphoma. Alterations including amplifications in PIK3CA, the gene encoding PI3K

α

, have been reported in nearly 70% of patients with mantle cell lymphoma (MCL)20 _{and in 6% of patients with CLL}21_{. In MCL cell lines and in patient}

samples, PI3K

α

expression increases significantly upon relapse during idelalisib treatment and dual inhibition of PI3K

α

and PI3K

δ

has been shown to be more efficacious in samples from relapsed MCL patients22_{. The dual PI3K}

γ

_/

δ

_{inhibitor duvelisib (IPI-145), the pan-PI3K}

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

inhibitors copanlisib (BAY80-6946) and buparlisb (BKM120), and the PI3K/mTOR inhibitor SAR245409 (voxtalisib, XL765) are currently under evaluation in CLL and NHL patients23-26_.

In this study, we investigated the role of PI3K

α

and PI3K

δ

inhibition in primary CLL cells by using the pan-PI3K/mTOR inhibitor SAR24540927_{, the pan-PI3K inhibitor SAR245408}28_,

the PI3K

α

inhibitor alpelisib (BYL719)29_{, and the PI3K}

δ

_{inhibitor idelalisib. We evaluated}

the impact of these inhibitors on PI3K/mTOR signaling, induction of apoptosis, cell adhesion and CD40-induced survival and proliferation in primary patient derived CLL cells. We also compared their impact on the proliferation and activation status of healthy T cells.

METHODS

Patient samples

Peripheral blood mononuclear cells (PBMC) of patients diagnosed with CLL (Supplemental table 1), obtained after Ficoll density gradient centrifugation (Pharmacia Biotech, Roosendaal, The Netherlands) were cryopreserved as previously described30_.

The study was approved by the medical ethics committee at the Academic Medical Center and written informed consent was obtained in accordance with the Declaration of Helsinki. Expression of CD5 and CD19 (both Beckton Dickinson (BD) Biosciences, San Jose, CA, USA) on leukemic cells was assessed by flow cytometry (FACScanto; BD Biosciences). CLL samples included in this study contained 81-99% CD5+_/CD19+ _cells.

PBMCs were isolated from buffy coats of healthy donors, aged between 18 and 64 years, from Sanquin Blood Supply (Amsterdam, the Netherlands) and cryopreserved in liquid nitrogen until use.

FISH and Gene mutational analyses

Deletions at the 11q22-q23 (ATM), 17p13 (TP53) and 13q14 loci and trisomy of chromosome 12 were detected by FISH by using locus-specific probes (Abott Vysis Inc). DNA was extracted by using the QiAamp DNA Blood Mini kit (Invitrogen) according to the manufacturer’s instructions. TP53 mutational analysis was either performed by a 454-based next generation sequencing (NGS) approach (Junior 454 platform, Roche, Penzberg, Germany) or using Sanger sequencing (exons 4-10)31_{. Primer sequences and}

technical details are available upon request. Mutation analysis of ATM (exons 1-62) was performed by Sanger sequencing as described previously32,33_.

Inhibitors and reagents

The pan-PI3K inhibitor pilaralisib (SAR245408, XL147, referred to in this study as SAR408), the PI3K/mTOR inhibitor voxtalisib (SAR245409, XL765, referred to in this study as SAR409), and the PI3K

α

inhibitor alpelisib (BYL719) were synthesized by Sanofi. The PI3K

δ

specific inhibitor idelalisib was obtained from Selleckchem (Houston, TX, USA). N-acetylcysteine (NAC) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The pan-caspase inhibitor QVD was purchased from R&D systems (Minneapolis, USA).

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

Cell culture and detection of apoptosis

CLL cells were thawed and incubated with different concentrations of drugs for 24, 48 or 72 hours. Where indicated, CLL cells were co-cultured in the presence/absence of 20 μM of the pan-caspase inhibitor QVD or 5 mM N-acetyl-L-cysteine (NAC). Viability was measured by DiOC6/PI staining as previously described30_{. Specific apoptosis was defined}

as [% cell death in treated cells] – [% cell death in medium control] / [% viable cells medium control] x 100.

To mimic the microenvironment, CLL cells were stimulated by coculture with NIH3T3 fibroblasts stably transfected with human CD40L (3T40L) or negative control plasmid (3T3) as described30 _{and co-cultured in the presence/absence of drugs at 1 μM or}

the indicated concentrations.

Adhesion assay

Cells were stimulated with either 200 ng/ml goat (Fab’)2 anti-human IgM (Sanbio, Uden, The Netherlands) or 50 ng/ml PMA (Sigma, Zwijndrecht, the Netherlands) for 30 min and adhesion to fibronectin coated plates was measured as described previously34_.

Migration assay

Transwell plates (pore size 5µm, Costar, Sigma) were coated with 1 µg/ml VCAM-1 (R&D systems, Minneapolis, MN

, USA)

and CLL cells were allowed to migrate to the lower compartment containing 100 ng/ml CXCL12 (Buchem BV, Apeldoorn, the Netherlands). The number of viable migrated cells was determined by FACS as described previously34_.

Proliferation assay

CLL cells were labelled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Life Technologies, Bleiswijk, The Netherlands) as described before35_{. Cells were cultured on 3T40L cells in presence of rhIL-21 (25ng/ml, Gibco, Life}

Technologies), with or without 1 μM of drugs. After 4 days, proliferation was assessed by FACSCalibur flow cytometer and analyzed with FlowJo software.

Western blot analysis

Western blot analysis was performed using standard techniques30_{.Membranes were}

probed with anti-pS6 (S240/244) (#5364), S6 (#2317), pAKT (T308) (#9275), pAKT (S473) (#4060), pErk (T202/204) (#9101), Erk (#9102) (Cell Signaling, Boston, MA, USA), and Bim (#SPC-113D) (Stressmarq, Victoria, Canada).

β

-actin (#sc-1616) (Santa Cruz Biotechnology, Dallas, TX, USA) was used as loading control.

Flow cytometry

Healthy PBMCs were stimulated with anti-CD3 (1xE, ascitus) and anti-CD28 (15E8; 5µg/ml). After 3 days PBMCs were resuspended in PBS, containing 0.5%(w/v) BSA and 0.01% sodium azide. PBMCs were incubated with saturating concentrations of CD3-AF700 (#826118C, Invitrogen), CD4-PE-Cy7 (#348809), CD8- PerCP-Cy5.5 (#341050), CD25-APC (#340907

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

(BD-biosciences). Flow cytometry measurements were performed on a FACSCanto using FACSDiva Software (BD Biosciences).

ELISA

Supernatants of the healthy PMCS were tested for cytokine production using eBioscience ELISAs for IFN-

γ

(#88-7316-88) and IL-13 (#88-7439-88). ELISA was performed according to the manufacturer’s directions.

Statistics and calculation of synergistic and additive effects

The paired Wilcoxon signed rank test was used to determine the significance of differences between two mean values. The one sample T test was used to determine the significance of differences between means and normalized values (100%). The one-way ANOVA was used to analyze differences between groups. * p <0,05;** p<0,01; *** p<0,001.

RESULTS

A pan-PI3K inhibitor inhibits the PI3K pathway in unstimulated primary CLL

cells and induces caspase-dependent cell death, irrespective of the ATM/

p53 status

Constitutive activation of the PI3K pathway in CLL cells has been reported9,10_{. We}

examined the activation of downstream effectors of the PI3K pathway in unstimulated CLL samples. Phosphorylation of AKT(T308), a proximal biomarker of PI3K activity was variable in CLL samples (n=8) (Figure 1A, Supplemental table 1). Similar results were found for pAKT(S473). Phosphorylation of S6(S240/244), a downstream biomarker of mTOR activation, was detected in all 8 CLL samples, with relatively little variation among patients (Figure 1A). In healthy donors, phosphorylation of S6(S240/244) was detected in B cells, but not in T cells (Figure 1B and Supplemental Figure 1). Phosphorylation of S6 was also detected in freshly isolated CLL cells (Figure 1C). Next, we compared the effect of BYL719 and idelalisib to SAR409 on baseline PI3K pathway activity in thawed primary CLL cells. BYL719 and idelalisib are potent and selective inhibitors of PI3Kα and PI3Kα, respectively (Supplemental Table 2). SAR409 is a potent inhibitor of all four class I PI3Ks and a weak inhibitor of mTOR in biochemical and cellular assays27 _{(Supplemental table}

2 and 3). Treatment with SAR409 resulted in strong reduction of S6 phosphorylation (Figure 1D) while inhibition with BYL719 or idelalisib partly reduced S6 phosphorylation. Similar reduction of S6 phosphorylation by SAR409 was found in freshly isolated CLL samples (Figure 1C). In BCR-stimulated CLL cells, phosphorylation of AKT(S473) was blocked completely by all three PI3K inhibitors, while only SAR409 was able to completely reduce S6 phosphorylation, (Supplemental Figure 2A-B). Treatment with SAR409 led to a time and dose-dependent induction of cell death of unstimulated primary CLL cells with a maximum impact at 48 hours with an IC50 of 0.86µM (Supplemental Figure 3A). In contrast, treatment with BYL719 and idelalisib induced cytotoxicity with an IC50 >10µM (Figure 2A). No differences were observed for SAR409-induced cell death between samples from previously treated and untreated CLL patients (Supplemental Figure 3B). The cytotoxic

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

Figure 1/Thijssen et al C LL pt #6 C LL pt #7A C LL pt #8 B ce ll H D # 1 B ce ll H D # 2 B ce ll H D # 3 T ce ll H D # 1 T ce ll H D # 2 T ce ll H D # 4 pS6 (S240/244) actin S6 Medi um SAR4 09 BYL7 19 Idela lisib 0.0 0.5 1.0 1.5 ** (n=4) p -S 6/ β -a cti n pS6 (S240/244) S6 A B C C LL pt #62 C LL pt #63 C LL pt #64 C LL pt #65 SAR409 - + - + - + - + pS6 (S240/244) actin D pAKT (T308) pS6 (S240/244) actin C LL pt #1C C LL pt #2A C LL pt #3 C LL pt #4A C LL pt #5 C LL pt #6 C LL pt #7A C LL pt #8 S6 pAKT (S473)

Figure 1. SAR409 inhibits the PI3K pathway in unstimulated primary CLL cells. A) Frozen CLL cells from 8 patients (pt#1-8) were thawed and cultured for 30 min. Protein lysates of CLL cells were probed for pS6(S240/244), S6, pAKT(S473), pAKT(T308) and actin for loading control. B) Protein lysates of CLL cells from 3 patients (pt #6-8) and purified B and T cells from 3 healthy donors were probed for pS6(S240/244), S6 and actin for loading control. C) Freshly isolated CLL cells from 4 patients (pt#62-65) were cultured in the presence or absence of 1 µM SAR409 for 2 hours. Protein lysates were probed for pS6 and actin for loading control. D) CLL cells were cultured in the presence or absence of 1 µM SAR409, BYL719 or Idelalisib for 2 hours. Protein lysates were probed for pS6, S6 and actin for loading control. Blot from one representative CLL sample is shown of four analyzed (pt#3,4A,9,10). Densitometric analysis of pS6 is shown. Bars represent the mean ± SEM, **p<0.01 (paired one sample T test).

effect of SAR409 was observed in all prognostic subgroups tested, including double 11q-/

ATM mutated, double 17p-/TP53 mutated, IgVH mutated and IgVH unmutated leukemia

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

Figure 2/Thijssen et al

A

B

0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 SAR409 BYL719 Idelalisib * ***** Drug concentration (µM) Sp eci fic ap op to si s ( % ) 0 0.01 0.1 1 10 0 20 40 60 80 100 _{IgVH mutated} IgVH unmutated ATM mutated TP53 mutated SAR409 (µM) Sp eci fic ap op to si s ( % )

C

0 0.01 0.1 1 10 0 20 40 60 80 100 Medium 20 µM QVD 5 mM NAC * * * SAR409 (µM) Sp eci fic ap op to si s ( % )

Figure 2. SAR409 induces apoptosis in unstimulated CLL cells from patients of 4 distinct prognostic groups. A) CLL cells were incubated with 0.001-10 μM SAR409, BYL719 or Idelalisib for 48 hours. Viability was assessed by DiOC6/PI staining and specific apoptosis was calculated (material and methods). Results are shown as mean ± SEM. (n=23, patient#7B, 9-27) *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA). B) CLL cells of patients of 4 clinical important prognostic subgroups (IgVH mutated (n=13, pt#7B, 9, 11-21), IgVH unmutated (n=10, pt#10A,10C,22-28), 11q-/ATM mutated (n=10 pt#34-44) and 17p-/TP53 mutated (n=6, pt#28-33) were incubated with SAR409 for 48 hours and specific apoptosis is shown. Results are shown as mean ± SEM. ns (one-way ANOVA). C) CLL cells were cultured with 20 µM QVD or 5 mM NAC and with increasing concentrations of SAR409 for 48 hours. Results are shown as mean ± SEM (n=4, pt#9,10A,21,56) *p<0.05 (one-way ANOVA).

in cell death induction was examined. As shown in Figure 2C, the pan-caspase inhibitor QVD completely blocked cytotoxicity of SAR409, whereas the ROS scavenger N-acetyl-cysteine (NAC) had no effect. In control experiments, NAC was able to inhibit CLL cell death induced by Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a potent uncoupler of oxidative phosphorylation and inducer of ROS (Supplemental Figure 4). In conclusion,

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

SAR409 induces caspase-dependent apoptosis in primary CLL cells irrespective of the p53/ ATM status.

BCR-controlled adhesion is dependent on PI3K

δ

activity and is inhibited

by SAR409 and idelalisib

Since targeting BCR-controlled integrin-mediated retention of malignant cells within their protective LN microenvironment is of major importance for the clinical efficacy of ibrutinib and idelalisib15,34_{, we evaluated the effect of inhibiting PI3K}

α

_{, PI3K}

δ

_{or all 4}

PI3K isoforms on BCR-controlled adhesion. First, the impact of PI3K inhibition on integrin-mediated adhesion to fibronectin was evaluated using the mantle cell lymphoma cell line JeKo-1, in which adhesion is strongly induced by BCR stimulation15,34_{. Significant}

inhibition of adhesion was observed with both SAR409 and idelalisib but not with BYL719,

Figure 3/Thijssen et al

A

B

M ed iu m M ed iu m SAR4 09 BYL 71 9 Id ela lis ib 0 50 100 αIgM **** R el at ive ad hesi on (% ) M ed iu m M ed iu m SAR4 09 BYL 71 9 Id ela lis ib 0 50 100 CXCL12 * R el at ive m ig rat io n ( % )

Figure 3. BCR-controlled adhesion and chemokine-mediated migration are inhibited by SAR409. A) CLL cells pretreated with 1 µM SAR409, BYL719 or Idelalisib were stimulated with

α

IgM and allowed to adhere to fibronectin-coated surfaces (n=4, pt#3,10B,6,23A). Graphs are presented as normalized mean ± SEM (100% = stimulated cells without inhibitors). **p<0.01 (paired one sample T test). B) CLL cells pretreated with 1 µM SAR409, BYL719 or Idelalisib were allowed to migrate toward CXCL12 on VCAM-1-coated transwell plates (n=3, pt#3,10B, 23A). Graphs are presented as normalized mean ± SEM (100% = stimulated cells without inhibitors). *p<0.05 (paired one sample T test).

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

Figure 4/Thijssen et al

Mediu m SAR4 09 BYL7 19 Idelal isib 0 20 40 60 80 100 CD40L activation ** * Sp eci fic su rvi val (% )

B

A

3T3 Mediu m SAR4 09 BYL 719 Idelal isib 0 1 2 3 4 *** CD40L activation R el at iv e exp re ss io n o f B IM ( % )

C

CD40L stimulation 3T3

SAR409 Idela BYL719

actin Bim 3T3 mediu m SAR4 09 BYL7 19 Idelal isib 0 1 2 3 4 CD40L activation ** ** B im /a cti n

D

Figure 4. SAR409 inhibits CD40-induced survival. CLL cells were cultured on fibroblasts (3T3) or CD40L-expressing fibroblasts (CD40L activation) in the presence or absence (medium) of 1 µM of SAR409, BYL719 or Idelalisib for 3 days. A) Survival was analyzed by DiOC6 staining. Results are shown as mean ± SEM. *p<0.05, **p<0.01; (one-way ANOVA) (n=10 pt#1B,7B,9,10A,16,23B,45-48). B) mRNA levels of Bim levels were measured by RT-MLPA (n=4 pt#4A, 46,49, 50). Results are shown as mean ± SEM. *p<0.05 (one-way ANOVA). C) Protein lysates were probed for Bim and actin for loading control. Blot from one representative CLL sample is shown of four analyzed (pt#1B,5,10A,45). D) Densitometric analysis of Bim is shown. Bars represent the mean ± SEM, * p<0.05 ** p<0.01.

suggesting that this process is dependent on PI3K

δ

activity (Supplemental Figure 5). As expected, integrin-mediated adhesion induced by PMA34_{, which activates protein kinase}

C independent of PI3K, was not affected (data not shown).

Next, we evaluated the impact of PI3K inhibition on BCR-controlled adhesion of primary CLL cells (Figure 3A). Only IgVH unmutated CLL cells were evaluated, as most IgVH mutated CLL cells are anergic to BCR-mediated adhesion34,39_{. Both SAR409 and}

idelalisib inhibited BCR-controlled adhesion, suggesting a key role for PI3K

δ

in this process in primary CLL as well.

Furthermore, we studied the effect of the PI3K inhibitors on chemokine-controlled migration of CLL cells towards CXCL12. Migration was partially inhibited by SAR409 (Figure 3B).

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

SAR409 inhibits CD40-mediated survival and suppression of Bim

We have previously shown that prolonged in vitro stimulation with CD40L leads to activation of NF-

κ

B mediated pro-survival signalling and to upregulation of activation markers40,41_{. CD40 mediated activation of the PI3K/AKT/mTOR pathway which was blocked}

by SAR409 and to some extent by BYL719, and in contrast was not affected by idelalisib (Supplemental Figure 6).

Activation of CLL cells by CD40 resulted in increased survival compared to control cells (Figure 4A) due to the differential expression of both pro- and anti-apoptotic Bcl-2 protein family members30,41,42_{. This pro-survival effect was significantly inhibited by SAR409}

and idelalisib (Figure 4A). The impact of SAR409 on the Bcl-2 protein family regulators of apoptosis was evaluated (Supplemental Figure 7). CD40 activation led to a decrease of the pro-apoptotic regulator BIM, via ERK signalling30 _{at the RNA and protein level}

(Figure 4B, C) and SAR409 repressed the CD40-mediated reduction of BIM expression (Figure 4B, C, D).

Figure 5/Thijssen et al

A

CD40 L Mediu m SAR4 09 BYL7 19 Idelal isib 0.0 0.2 0.4 0.6 *** *** CD40L + IL-21 D iv is io n in de x

B

Control SAR409

Figure 5. PI3K inhibitors inhibit proliferation of CLL cells. A) CFSE labelled CLL cells were cultured on fibroblast expressing CD40L with IL-21 (control/grey line) and co-treated with 1 µM SAR409 (black line). After 4 days, CFSE was measured by FACS. B) Division index was calculated with FlowJo program. Results are shown as mean ± SEM (n=11, pt#2B, 3,24A,44,51-55). ***p<0.001 (Wilcoxon matched pairs test).

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

Figure 6/Thijssen et al

A

B

T cells 0 0.01 0.1 1 10 0 20 40 60 80 100 BYL719 Idelalisib SAR409 Drug concentration (µM) Sp eci fic ap op to si s ( % ) B cells 0 0.01 0.1 1 10 0 20 40 60 80 100 SAR409 BYL719 Idelalisib Drug concentration (µM) Sp eci fic ap op to si s ( % ) Contr ol SAR4 09 BYL7 19 Idelal isib 0 100000 200000 300000 400000 500000 pg of IFN -γ contr ol SAR4 09 BYL7 19 Idelal isib 0 500 1000 1500 2000 2500 * pg of IL-13

C

D

CD8+ _{T cells} contr ol SAR4 09 BYL7 19 Idelal isib 0 20 40 60 80 100 % d iv id ed c ells CD4+ _{T cells} contr ol SAR4 09 BYL7 19 Idelal isib 0 20 40 60 80 100 ***** *** % d iv id ed c ells

E

F

G

H

CD8+ _{T cells} Control SAR409 BYL719 Idelalisib CD4+ _{T cells} Control SAR409 BYL719 Idelalisib

Figure 6. PI3K inhibitors do not cause cytotoxicity in T cells but inhibit proliferation and alter cytokine production in healthy T cells. A-B) PBMCs from healthy donors were incubated with 0.01-10 μM SAR409, BYL719 or Idelalisib for 48 hours. Specific apoptosis was analyzed in CD19+ B cells (A) or in CD3+ T cells (B). Results are shown as mean ± SEM, n=3. C-H) PBMCs from healthy donors were stimulated with CD3/CD28 in the presence or absence of 1 μM SAR409, BYL719 or Idelalisib for 72 hours (n=3). C-D) CD25 expression was measured by FACS in CD8+ T cells (C) or CD4+ T cells (D) treated with SAR409 (dark black line), BYL719 (grey line) and Idelalisib (dotted line) or control (black line). Isotype is shown in grey. One histogram is shown of three donors analyzed. E) IFN-

γ

production by the T-cells was measured by ELISA. Bars represent the mean ± SEM, ns (one-way ANOVA) F) IL-13 by the T-cells was measured by ELISA. Bars represent the mean ± SEM, * p<0.05 (one-way ANOVA) G) CFSE was measured by FACS in CD8+ T cells. Bars represent the mean ± SEM, ns (one-way ANOVA) H) CFSE was measured by FACS in CD4+ T cells. Bars represent the mean ± SEM, **p<0.01, ***p<0.001 (one-way ANOVA).

SAR245409 BLOCKS CRITICAL SIGNALLING P A THW A YS IN CLL

2

PI3K blockade inhibits BCR-independent proliferation of CLL cells

BCR-independent proliferation can be mediated by cytokines such as IL-21 produced by LN resident activated T cells and follicular helper T cells35_{. As shown in Figure 5A and B,}

combination of CD40 activation with IL-21 treatment led to cell proliferation and this was partially inhibited by all three inhibitors.

PI3K inhibition inhibits cytokine production by activated T cells

We evaluated the impact of SAR409 on B and T cells derived from healthy donors (HD). Although SAR409 had a pro-apoptotic effect on normal B cells, the IC50 value (4.26µM) was 5-fold higher than the IC50 value in CLL cells (0.86µM) (Figure 6A). BYL719 and idelalisib also had a similar weak impact on B cells with an IC50 > 10µM (Figure 6A). No cytotoxic impact was observed on T cells from HD or from CLL patients with the PI3K inhibitors (Figure 6B and Supplemental Figure 8). PI3K inhibition also had no impact on CD3 and CD28-mediated CD25 expression, a marker for activation, in HD derived T cells (Figure 6C-D). A trend of inhibition of IL-13 and IFN-

γ

production by the T-cells was observed with all 3 inhibitors (Figure 6E-F). Only SAR409 completely blocked IL-13 production (Figure 6F). SAR409 partially inhibited the proliferation of CD8+ _{T cells and}

almost completely blocked the proliferation of CD4+ _{T cells (Figure 6G-H). BYL719 and}

idelalisib partially inhibited the proliferation of both CD8+ _{and CD4}+ _{T cells (Figure 6G-H).}

The pan-PI3K inhibitor SAR408 has similar activity in CLL cells as SAR409

Recently, promising preliminary clinical efficacy data of the pan-PI3K inhibitor SAR408 (SAR245408, pilaralisib, XL147) in CLL and lymphoma patients were reported28_{. As}

the kinase inhibition profile of SAR408 closely overlaps with SAR409 (Supplemental table 2), we compared the impact of SAR408 and SAR409 on primary CLL cells in vitro. To avoid loss of activity due to the high protein binding of SAR408, the impact of SAR408 on the PI3K pathway, cytotoxicity and cell adhesion was evaluated in medium containing low serum (0.1%). Phosphorylation of S6 was completely reduced by SAR408 in CLL cells at comparable levels to SAR409 (Figure 7A-B). SAR408 also induced apoptosis in CLL cells at levels similar to SAR409 (Figure 7C). Finally, SAR408 significantly inhibited BCR-induced adhesion of the CLL cells and the JeKo-1 cell line to fibronectin (Figure 7D and Supplemental Figure 9). These data demonstrate that SAR408 exhibits effects on CLL cells comparable to SAR409.

DISCUSSION

Our study reveals that a pan-PI3K inhibitor is more cytotoxic to CLL cells than PI3K

α

or PI3K

δ

isoform selective inhibitors. Furthermore, combined inhibition of several PI3K isoforms can block signaling pathways that are critical for CLL survival, adhesion and proliferation in the LN microenvironment.

Idelalisib is currently approved for relapsed/refractory CLL in combination with rituximab and has shown impressive clinical activity43,44 _{due to inhibition of the PI3K}

δ

_-mediated