The interplay between microenvironmental signaling and novel targeted drugs in CLL - Chapter 2: The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival,

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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

Link to publication

Citation for published version (APA):

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

drugs in CLL.

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THE PAN PHOSPHOINOSITIDE 3-KINASE/

MAMMALIAN TARGET OF RAPAMYCIN

INHIBITOR SAR245409 (VOXTALISIB/XL765)

BLOCKS SURVIVAL, ADHESION

AND PROLIFERATION OF PRIMARY CHRONIC

LYMPHOCYTIC LEUKEMIA CELLS

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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.

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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_deliver_{critical 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

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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).

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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

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(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

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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

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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,

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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,

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).

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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).

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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).

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).

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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).

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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

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2

environment13,14_{. Yet, almost half (44-48%) of relapsed/refractory CLL patients progressed}

at 12 months44,45_{. In this study, we show that inhibition of either PI3K}

α

_{or PI3K}

δ

_isoforms

only promotes limited cell death, resulting in residual disease and the risk of development

of resistant clones. Simultaneous inhibition of PI3K

γ

and PI3K

δ

by duvelisib (IPI-145) did

not result in increased cytotoxicity46,47_{. We show that in primary CLL cells a downstream}

target of the PI3K/mTOR pathway, S6, is constitutively phosphorylated, demonstrating constitutive activation of the PI3K/mTOR pathway in the absence of BCR activation and thus providing rationale for therapeutic targeting of this pathway. pS6 was not fully blocked

by inhibition of either the

α

or

δ

PI3K isoform, demonstrating the existence of redundancy

mechanisms in CLL cells. Fully blocking the PI3K/mTOR pathway by a pan-PI3K inhibitor exerts prominent caspase-dependent cytotoxicity in CLL cells (Table 1). Importantly, 1 µM SAR409 induced ±50% apoptosis in p53 and ATM dysfunctional CLL samples. SAR409 was also able to inhibit the CD40-mediated survival in CLL cells. However, inhibition of

PI3K

α

and

β

in healthy tissues might also decrease the tolerability of these drugs. It has

been shown that the pan-PI3K inhibitor SAR408/pilaralisib has promising clinical benefit in CLL patients, with neutropenia, diarrhea, and anemia occurring at rates similar to those

Figure 7. SAR408 has similar in vitro activity in CLL cells as SAR409. A) CLL cells were thawed and cultured in the presence or absence of 1 µM of inhibitors for 2 hours. Protein lysates were probed for pS6 and actin for loading control. Blot from one representative CLL sample is shown of three analyzed. B) Densitometric analysis of pS6 is shown. Bars represent the mean ± SEM, ** p<0.01 (paired one sample T test) (n=3 pt#45,49,50). C) CLL cells were incubated with 1 μM SAR408 or SAR409 (n=9, pt# 1C, 12B,13, 56-61) and assayed for apoptosis. D) CLL cells pretreated with 1 µM SAR409 or SAR408 were stimulated with

α

IgM and allowed to adhere to fibronectin-coated surfaces (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).

A

Figure 7/Thijssen et al

B

contr ol SAR4 08 SAR4 09 BYL7 19 Idelal isib 0.0 0.5 1.0 1.5 ***** p-S6/ β-a cti n SAR408 SAR409 0 20 40 60 80 100 sp eci fic ap op to si s ( % )

C

D

Control pS6 (S240/244) actin CLL pt #49

SAR408 SAR409 BYL719 Idelalisib

Mediu m Mediu m SAR4 08 SAR4 09 0 50 100 αIgM * R el at ive ad hesi on (% )

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2

lymphocytosis was observed, which might be due to increased levels of apoptosis induced

by pilaralisib28_.

Recently, other PI3K inhibitors have been evaluated in vitro in primary CLL samples.

The PI3K

δ

/

γ

inhibitor IPI-145 and the pan-PI3K inhibitor BKM120 showed disruption of

antigen- and stromal cell-mediated survival49,50_{. In addition, IPI-145 and BKM120 impaired}

chemotaxis of CLL cells, which was also observed for the pan-PI3K inhibitor copanlisib47,49,50_.

Although the pan-PI3K inhibitors seem to increase apoptosis as compared to the selective PI3K isoform inhibitors in primary CLL cells, the level of cytotoxicity varies between

drugs46-51_{. So far, drug-induced effects on proliferation and BCR mediated adhesion have}

not been studied with these compounds.

Concerns have been raised about inhibiting PI3K

δ

in leukemia as this might also inhibit

normal immune cells, including T cells. Our data show that the PI3K inhibitors did not induce cytotoxicity or inhibit the activation of T cells. However, all of the tested PI3K

inhibitors inhibited proliferation of CD4+_{T cells, which was most prominent in SAR409}

treated cells. It has been recently reported that PI3K

δ

inhibition reduces CD4+_CD25+

regulatory T cell (Treg)-mediated suppression of cancer immune surveillance52, 53, 54,55_.

Cytokines produced by activated T cells, including IFN-

γ

and IL-13 have been reported

to induce pro-survival signals in CLL cells56-58_{. All three PI3K inhibitors inhibited IFN-}

γ

_and

IL-13 production, which may shift the balance to a less tumor supportive microenvironment. However, cytokine production also plays a pivotal role in the protection against infections. The most frequent adverse events seen in clinical studies with idelalisib are pneumonia and

diarrhoea43,44_{which may be associated with the inhibition of cytokines and/or the inhibition}

of proliferation of CD4+_{T cells, including Tregs.}

Clinical evaluation of the pan-PI3K inhibitors copanlisib and buparlisib is ongoing in CLL patients. Whether pan-PI3K inhibition might prevent emergence of resistance resulting in prolonged progression free survival, or whether it might be effective in patients who have failed idelalisib, is as yet unknown. Further clinical evaluation, especially in CLL patients harboring a TP53 or ATM mutation, either as monotherapy or in combination, seems warranted.

ACKNOWLEDGEMENTS

We thank the CLL patients for their blood donations and Marjolein Spiering and Dieuwertje Luijks for database management and CLL cell phenotyping. APK is a recipient of a Dutch Cancer Society Clinical Fellowship.

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2 REFERENCES

1. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352(8):804-815. 2. Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand AV. Human bone marrow stromal

cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro. Br J Haematol. 1996;92(1):97-103.

3. Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell’Aquila M, Kipps TJ. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood. 2000;96(8):2655-2663.

4. Ghia P, Strola G, Granziero L, et al. Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4+, CD40L+ T cells by producing CCL22. Eur J Immunol. 2002;32(5):1403-1413.

5. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7(8):606-619.

6. Chantry D, Vojtek A, Kashishian A, et al. p110delta, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes. J Biol Chem. 1997;272(31):19236-19241.

7. Reif K, Okkenhaug K, Sasaki T, Penninger JM, Vanhaesebroeck B, Cyster JG. Cutting edge: differential roles for phosphoinositide 3-kinases, p110gamma and p110delta, in lymphocyte chemotaxis and homing. J Immunol. 2004;173(4):2236-2240.

8. Beer-Hammer S, Zebedin E, von Holleben M, et al. The catalytic PI3K isoforms p110gamma and p110delta contribute to B cell development and maintenance, transformation, and proliferation. J Leukoc Biol. 2010;87(6):1083-1095.

9. Herman SE, Gordon AL, Wagner AJ, et al. Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood. 2010;116(12):2078-2088.

10. Ringshausen I, Schneller F, Bogner C, et al. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cdelta. Blood. 2002;100(10):3741-3748.

11. Srinivasan L, Sasaki Y, Calado DP, et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell. 2009;139(3):573-586.

12. Clayton E, Bardi G, Bell SE, et al. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation. J Exp Med. 2002;196(6):753-763.

13. Fiorcari S, Brown WS, McIntyre BW, et al. The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells. PLoS One. 2013;8(12):e83830.

14. 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.

15. de Rooij MF, Kuil A, Kater AP, Kersten MJ, Pals ST, Spaargaren M. Ibrutinib and idelalisib synergistically target BCR-controlled adhesion in MCL and CLL: a rationale for combination therapy. Blood. 2015;125(14):2306-2309.

16. 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.

17. Juric D, Castel P, Griffith M, et al. Convergent loss of PTEN leads to clinical resistance to a PI(3) Kalpha inhibitor. Nature. 2015;518(7538):240-244.

(18)

2

19. Ramadani F, Bolland DJ, Garcon F, et al. The PI3K isoforms p110alpha and p110delta are essential for pre-B cell receptor signaling and B cell development. Sci Signal. 2010;3(134):ra60.

20. Psyrri A, Papageorgiou S, Liakata E, et al. Phosphatidylinositol 3’-kinase catalytic subunit alpha gene amplification contributes to the pathogenesis of mantle cell lymphoma. Clin Cancer Res. 2009;15(18):5724-5732.

21. Brown JR, Hanna M, Tesar B, et al. Integrative genomic analysis implicates gain of PIK3CA at 3q26 and MYC at 8q24 in chronic lymphocytic leukemia. Clin Cancer Res. 2012;18(14):3791-3802. 22. Iyengar S, Clear A, Bodor C, et al. P110alpha-mediated constitutive PI3K signaling limits

the efficacy of p110delta-selective inhibition in mantle cell lymphoma, particularly with multiple relapse. Blood. 2013;121(12):2274-2284.

23. IPI-145 shows promise in CLL patients. Cancer Discov. 2014;4(2):136.

24. Dreyling M, Cunningham D, Bouabdallah K, et al. Phase 2A Study of Copanlisib, a Novel PI3K Inhibitor, in Patients with Indolent Lymphoma. Blood. 2014;124(21).

25. Daver N, Kantarjian H, DeBose L, et al. Buparlisib, a Pi3k Inhibitor, Demonstrates Acceptable Tolerability and Preliminary Activity in a Phase I/Ii Trial of Patients with Advanced Leukemias. Haematologica. 2014;99:36-36.

26. Papadopoulos KP, Egile C, Ruiz-Soto R, et al. Efficacy, safety, pharmacokinetics and pharmacodynamics of SAR245409 (voxtalisib, XL765), an orally administered phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor: a phase 1 expansion cohort in patients with relapsed or refractory lymphoma. Leuk Lymphoma. 2014:1-8.

27. Yu P, Laird AD, Du X, et al. Characterization of the activity of the PI3K/mTOR inhibitor XL765 (SAR245409) in tumor models with diverse genetic alterations affecting the PI3K pathway. Mol Cancer Ther. 2014;13(5):1078-1091.

28. Brown JR, Davids MS, Rodon J, et al. Phase I trial of the pan-PI3K inhibitor pilaralisib (SAR245408/ XL147) in patients with chronic lymphocytic leukemia (CLL) or relapsed/refractory lymphoma. Clin Cancer Res. 2015.

29. Fritsch C, Huang A, Chatenay-Rivauday C, et al. Characterization of the novel and specific PI3Kalpha inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials. Mol Cancer Ther. 2014;13(5):1117-1129.

30. 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.

31. Te Raa GD, Derks IA, Navrkalova V, et al. The impact of SF3B1 mutations in CLL on the DNA-damage response. Leukemia. 2015;29(5):1133-1142.

32. Austen B, Powell JE, Alvi A, et al. Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. Blood. 2005;106(9):3175-3182.

33. Skowronska A, Parker A, Ahmed G, et al. Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial. J Clin Oncol. 2012;30(36):4524-4532.

34. 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.

(19)

2

35. 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.

36. Tonino SH, van Laar J, van Oers MH, Wang JY, Eldering E, Kater AP. ROS-mediated upregulation of Noxa overcomes chemoresistance in chronic lymphocytic leukemia. Oncogene. 2011;30(6):701-713. 37. Zhou Y, Hileman EO, Plunkett W, Keating MJ, Huang P. Free radical stress in chronic lymphocytic

leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents. Blood. 2003;101(10):4098-4104.

38. Trachootham D, Zhang H, Zhang W, et al. Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism. Blood. 2008;112(5):1912-1922.

39. Guarini A, Chiaretti S, Tavolaro S, et al. BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells. Blood. 2008;112(3):782-792.

40. Tromp JM, Tonino SH, Elias JA, et al. Dichotomy in NF-kappaB signaling and chemoresistance in immunoglobulin variable heavy-chain-mutated versus unmutated CLL cells upon CD40/TLR9 triggering. Oncogene. 2010;29(36):5071-5082.

41. Kater AP, Evers LM, Remmerswaal EB, et al. CD40 stimulation of B-cell chronic lymphocytic leukaemia cells enhances the anti-apoptotic profile, but also Bid expression and cells remain susceptible to autologous cytotoxic T-lymphocyte attack. Br J Haematol. 2004;127(4):404-415. 42. Smit LA, Hallaert DY, Spijker R, et al. Differential Noxa/Mcl-1 balance in peripheral

versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood. 2007;109(4):1660-1668.

43. 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.

44. 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. 45. Sharman JP CS, Furman RR, Cheson BD, JM Pagel, Hillmen P, et al. Second Interim Analysis of

a Phase 3 Study of Idelalisib (ZYDELIG®) Plus Rituximab (R) for Relapsed Chronic Lymphocytic Leukemia (CLL): Efficacy Analysis in Patient Subpopulations with Del(17p) and Other Adverse Prognostic Factors. In: Abstract ed. ASH annual meeting and exposition 2014.

46. Dong S, Guinn D, Dubovsky JA, et al. IPI-145 antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia cells. Blood. 2014;124(24):3583-3586.

47. Gockeritz E, Kerwien S, Baumann M, et al. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells. Int J Cancer. 2015.

48. O’Brien S, Patel M, Kahl BS, et al. Duvelisib (IPI-145), a PI3K-delta,gamma Inhibitor, Is Clinically Active in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia. Blood. 2014;124(21). 49. Balakrishnan K, Peluso M, Fu M, et al. The phosphoinositide-3-kinase (PI3K)-delta and gamma

inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL. Leukemia. 2015.

50. Rosich L, Saborit-Villarroya I, Lopez-Guerra M, et al. The phosphatidylinositol-3-kinase inhibitor NVP-BKM120 overcomes resistance signals derived from microenvironment by regulating the Akt/ FoxO3a/Bim axis in chronic lymphocytic leukemia cells. Haematologica. 2013;98(11):1739-1747. 51. Blunt MD, Carter MJ, Larrayoz M, et al. The PI3K/mTOR inhibitor PF-04691502 induces

apoptosis and inhibits microenvironmental signaling in CLL and the Emicro-TCL1 mouse model. Blood. 2015;125(26):4032-4041.

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2

53. Beyer M, Kochanek M, Darabi K, et al. Reduced frequencies and suppressive function of CD4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood. 2005;106(6):2018-2025.

54. Jak M, Mous R, Remmerswaal EB, et al. Enhanced formation and survival of CD4+ CD25hi Foxp3+ T-cells in chronic lymphocytic leukemia. Leuk Lymphoma. 2009;50(5):788-801.

55. Dasgupta A, Mahapatra M, Saxena R. Flow cytometric immunophenotyping of regulatory T cells in chronic lymphocytic leukemia: comparative assessment of various markers and use of novel antibody panel with CD127 as alternative to transcription factor FoxP3. Leukemia & Lymphoma. 2013;54(4):778-789.

56. Chaouchi N, Wallon C, Goujard C, et al. Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis. Blood. 1996;87(3):1022-1029. 57. Podhorecka M, Dmoszynska A, Rolinski J. Intracellular IFN-gamma expression by CD3+/CD8+ cell

subset in B-CLL patients correlates with stage of the disease. Eur J Haematol. 2004;73(1):29-35. 58. Zaki M, Douglas R, Patten N, et al. Disruption of the IFN-gamma cytokine network in chronic

lymphocytic leukemia contributes to resistance of leukemic B cells to apoptosis. Leuk Res. 2000;24(7):611-621.

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Ta b le S 1. P at ie nt s’ c ha ra ct er is tic s Patient Age Gender

IgVH mutational status

WBC 10^9/L % lymphocytes %CD19+/CD5+ cells FISH Tr eatment befor e sampling 1A 55 F U 226.1 93.1 93.9 none FCR 1B 55 F U 180 ND 95.1 none FCR 1C 55 F U 272 ND 95.4 none FCR 2a 66 M M ND ND 95.2 none none 2b 66 M M 93.4 ND 90.8 none none 3 70 M U 422 ND 92.8 17p-chlorambucil, CVP/COP , FCR 4A 63 F M 153,0 96,0 95.7 13q-, 17p-chlorambucil, FCR 4B 62 F M 173 ND 99.9 13q-, 17p-chlorambucil 5 61 F M 32.8 87 52.2 13q-none 6 80 F U 63 ND 85.9 ND chlorambucil 7A 83 F M 56.8 87.6 88.1 13q-, tris12 none 7B 83 F M 55.4 87 89.6 13q-, tris12 none 8 75 M U 127 ND 93 13q-none 9 56 F M ND ND 94.7 13q-none 10A 74 F U 89.9 ND 91.4 tris12 none 10B 74 F U 110 87.3 91.1 tris12 none 10C 73 F U 115.3 ND 90.3 tris12 none 11 67 F M 88.7 ND 86.9 13q-chlorambucil + pr ednison, fludarabine 12A 63 F M 32.2 86.7 80.6 13q-none 12B 66 F M 23.8 80 88.8 13q-none 13 62 F M 84.8 ND 99.9 13q-none 14 88 F M 53.8 87.2 87.1 ND none 15 78 F M 30.8 84.2 86.1 ND none 16 58 M M 64.3 ND 88.1 13q-none 17 61 F M 46.1 ND 91.1 13q-none 18 42 M M 66.6 92 86.3 13q-none

SUPPLEMENTARY MATERIALS

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2

Ta b le S 1. (co nt inu ed ) Patient Age Gender

WBC 10^9/L % lymphocytes %CD19+/CD5+ cells FISH Tr eatment befor e sampling 19 81 F M 23.1 87.3 93.3 ND none 20 46 M M 12.8 72.4 99.5 ND none 21 86 M M 90.5 90.8 81 13q-chlorambucil 22A 56 M U 78.1 ND 89.6 tris12 chlorambucil 22B 50 M U 149 94 90 tris12 chlorambucil 23A 60 M U ND ND 91 13q-none 23B 60 M U 69.5 ND 94 13q-none 24A 74 M U 166.6 ND 84.2 ND chlorambucil 24B 73 M U 116.8 ND 98.4 ND none 25 60 M U 21.9 75.1 99.2 ND none 26 76 M U 49.7 ND 88.6 13q-none 27 52 M U 132 92.1 86.2 tris12 none 28 36 M U 82.5 75.1 85.13 17p-CVP/COP , FCR, alemtuzumab, R-CHOP , R-DHAP 29 59 M U ND ND ND 17p-none 30 82 M M 137 94.8 83.5 13q-, 17p-chlorambucil, alemtuzumab 31 58 M M 155 ND 87.9 tris12, 17p-chlorambucil + pr ednison, R-CHOP FCR, Rituximab, R-DHAP 32 63 unknown U ND ND ND

17p-fludarabine + cyclophosphamide, R-CHOP

, alemtuzumab 33 75 unknown U ND ND ND 17p-, 11q-chlorambucil, FCR, alemtuzumab 34 53 M U 31.1 95.8 82.1 11q-chlorambucil, radiotherapy , CVP/COP FCR 35 72 M unknown 87.9 86 82.4 13q-, 11q-chlorambucil, R-CVP , FCR + R-CHOP dasatinib + fludarabine 36 44 M U ND ND ND 13q-, 11q-none

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2

WBC 10^9/L % lymphocytes %CD19+/CD5+ cells FISH Tr eatment befor e sampling 37 68 unknown U ND ND ND 11q-none 38 80 unknown M ND ND ND 11q-none 39 70 unknown U ND ND ND

11q-fludarabine + cyclophosphamide, fludarabine

40 77 unknown U ND ND ND 11q-none 41 68 unknown U ND ND ND 11q-none 42 44 unknown U ND ND ND 11q-none 43 64 unknown U ND ND ND

11q-fludarabine + cyclophosphamide, alemtuzumab

44 27 M U 160.5 94.6 93.8 11q-none 45 58 F M 170.9 ND 97.9 13q-, tris12

chlorambucil, fludarabine + cyclophosphamide, fludarabine

46 60 M M 56.5 90 95.2 ND none 47 63 F M 92.7 ND 93.7 none none 48 68 F U 71.7 83.2 97 ND none 49 67 F M 64.8 96.0 94.8 13q-chlorambucil+ pr ednisolone + cyclosporine 50 78 M U 100 74 94.1 tris12 none 51 80 M M 149.4 92 98.2 none chlorambucil 52 68 F U 100.5 89 84.8 tris12 none 53 73 M U 108 94.7 96.4

17p-chlorambucil, fludarabine, alemtuzumab, FCR

54 65 F M 59 92 87.6 ND none 55 50 F M 80.2 96 91.4 ND none 56 60 F U 265 ND 99.8 13q-FCR 57 41 M M 40 84 91.4 13q-none

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2

WBC 10^9/L % lymphocytes %CD19+/CD5+ cells FISH Tr eatment befor e sampling 58 58 M U 55.7 83 94.4

13q-chlorambucil + fludarabine + cyclofosfamide

59 60 M M 85.5 94 95.8 13q-none 60 65 F U 89.9 90 89 ND none 61 71 F M 47.3 91.1 95.9 ND none 62 66 M unknown 32.0 81.9 90.5 13q-none 63 56 M U 75.6 ND 64.6 13q-, 11q-none 64 71 F unknown 72.6 ND 45.2 ND none 65 78 F unknown 39.61 ND 47.5 ND none ND: not deter mined; A, B, C r efer to dif fer

ent sampling times of

the

same patient; FCR: fludarabine + cyclophosphamide + rituximab; CVP/COP: cyclophosphamide

+ vincristine/oncovin + pr

ednisolone

R-CHOP: rituximab + cyclophosphamide + vincristine/oncovin + pr

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2

Table S2. Biochemical Kinase Inhibition Profile of SAR409, SAR409, BYL719 and Idelalisib. The half maximal inhibitory concentration (IC50) for Class I PI3Ks and mTOR for compounds SAR409, SAR408, BYL719 and Idelalisib are reported in nM

IC₅₀ (nM)

Family Kinase SAR409 SAR2408 BYL7191 _Idelalisib2

PI3K PI3K

α

101 48 4.6 820 Class I PI3K

β

128 617 1156 565 PI3K

δ

91 10 290 2.5 Class I_B PI3K

γ

43 260 250 89 Class III VPS34 3754 6300 >9,100 978 PI3K-related mTORa ₁₆₁₃ _{> 15,000} _>9,100 _>1,000

IC50, concentration required for 50% target inhibition; mTORC1/2, mammalian target of rapamycin complex 1/2; PI3K, phosphatidylinositol 3-kinase; VPS34, vacuolar sorting protein 34.

a_{Kinase assay using recombinant mTOR enzyme}

1 _{Values derived from Fritch C, et al. Mol Cancer Ther, 2014, 13(5):1117-29.} 2_{Values derived from Lannutti BJ, et al. Blood, 2011, 117(2), 591-594.}

Table S3. Effects of SAR245409 (XL765) on Phosphorylation of AKT and S6 in Cell Models

Cell line Kinase pAKT IC50 (nM)

Myr-p110

β

3T3 MEF Myr-PI3K

β

53a

Myr-p110

δ

3T3 MEF Myr-PI3K

δ

36a

TSC2 (-/-) TP53 (-/-) MEF mTOR 4100b

IC50 values determined using a pAKT(S473) and b p4EBP(S65) ELISA/MSD assays.

Figure S1. CLL cells and healthy B cells express pS6. Frozen CLL cells from 6 patients (patient#4B, 7B, 22B, 47, 55, 60) or PBMCs from 6 healthy donors were thawed and cultured for 30 min. pS6 expression was analyzed in CD19+ cells or in CD3+ cells by FACS. Data were normalized for isotype control. *p<0.05.

Supplemental Figure 1/Thijssen et al

CLL T cell HD B cell H D 0 2 4 6 8 10 * * G M FI p-S6

(26)

2

Figure S2. SAR409 inhibits the PI3K pathway in BCR stimulated primary CLL cells. CLL cells pretreated with 1 µM SAR409, BYL719 or Idelalisib were stimulated with

α

IgM for 30 min. Protein lysates were probed for pAKT(S473), pS6 (S240/244), S6, pErk (T202/204), Erk and actin as loading control. B) Densitometric analysis of pAkt, pS6 and pErk are shown.

mediu m co ntrol mediu m SAR4 09 BYL71 9 Idelal isib 0 1 2 3 4 α-IgM stimulation p-Akt (S )/act in mediu m co ntrol mediu m SAR4 09 BYL71 9 Idelal isib 0.0 0.5 1.0 1.5 2.0 α-IgM stimulation p-S6/ act in mediu m co ntrol mediu m SAR4 09 BYL71 9 Idelal isib 0.0 0.5 1.0 1.5 2.0 α-IgM stimulation p-Er k/ act in pAKT (S473) pS6 (S240/244) pErk (T202/204) actin

Medium αIgM stimulation

SAR409 BYL719 Idelalisib

Erk S6

A

(27)

2

Figure S3. SAR409 induce dose-dependent cytotoxicity after 48 hours. A) CLL cells were incubated with 0.001-10 μM SAR409 for 24, 48 and 72 hours. Viability was assessed by DiOC6/PI staining and specific apoptosis was calculated (material and methods). Results are shown as mean ± SEM (n=5, patient#6, 10C, 12A, 13, 22A). B) Previously treated CLL cells (n=4 patient#11, 21, 22, 24) or previously untreated CLL cells (n=23 patient#7B, 9, 10, 12-20, 23, 25-27) were incubated with 0.001-10 μM SAR409 for 48 hours. Viability was assessed by DiOC6/PI staining and specific apoptosis was calculated. Results are shown as mean ± SEM.

Figure S4. CCCP-induced cell death is ROS dependent. CLL cells were cultured 5 mM NAC and with increasing concentrations of CCCP. Viability was analyzed by DiOC6/PI staining and specific apoptosis was calculated.

Supplemental Figure 3/Thijssen et al

0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 24hrs 48hrs 72hrs SAR409 µM Sp eci fic ap op to si s ( % ) 0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 Previously treated Previously untreated SAR409 (µM) Sp eci fic ap op to si s ( % )

A

B

Supplemental Figure 4/Thijssen et al

0 1 10 100 0 20 40 60 80 100 medium 5 mM NAC CCCP (µM) Sp eci fic ap op to si s ( % )

(28)

2

Figure S5. BCR-controlled adhesion of mantle cell lymphoma cell line JeKo-1 to fibronectin is inhibited by SAR409 and Idelalisib. JeKo-1 cells pretreated with 1 µM SAR409, BYL719 or Idelalisib were stimulated with

α

IgM and allowed to adhere to fibronectin-coated surfaces (n=5). Graphs are presented as normalized mean ± SEM (100% = stimulated cells without inhibitors). **p<0.01 (paired one sample T test).

Figure S6. SAR409 inhibits the PI3K pathway in CD40 stimulated primary CLL cells. CLL cells were cultured on fibroblasts (3T3) or CD40L-expressing fibroblasts (CD40L stimulation) in the presence or absence of 1uM of SAR409, BYL719 and Idelalisib for 3 days. Protein lysates were probed for pS6 (S240/244), pAkt (S473) and actin for loading control. Blot from one representative CLL sample is shown of three analyzed (pt#11A,23A,46).

Figure S7. SAR409 alter BIM expression. CLL cells were cultured on fibroblast expressing CD40L and co-treated with 1µM SAR409 for 3 days. mRNA levels of pro-/anti-apoptotic mediators in CLL cells were measured by RT-MLPA (n=4). Results are shown as mean ± SEM. *p<0.05 (one-way ANOVA).

M ed iu m M ed iu m SAR4 09 BYL 71 9 Id ela lis ib 0 50 100 αIgM ** ** JeKo-1 cells R el at ive ad hesi on (% )

Supplemental Figure 6/Thijssen et al

CD40L stimulation 3T3

actin

pS6 (S240/244) SAR409 Idelalisib BYL719

pAkt (S473)

Supplemental Figure 7/Thijssen et al

B cl-w _Bcl-x _Bcl-2 _Bfl-1 M cl-1 _Noxa PUM A B im _Bid H ar aki ri B mf Ma p1 _Bak _Bax Bc l-RAM BO cI AP1 CI AP2 _XIAP A po llo n p21 APAF 1 FL IP FL IP 2 OMI SM AC G ranz ym eB Pe rf or in _PI9 CD9 5 CD1 78 _Nix NI P3 0 5 10 15 20 25 _{3T3 - medium} 3T40L - medium

Bcl-2-like genes BH3-only genes Bax-like genes IAP's genes Apoptosis related genes

3T40L + 1 uM SAR409 * R el at ive E xp ressi on (% )

(29)

2

Figure S8. SAR409 does not cause cytotoxicity in T cells from CLL patients. PBMCs from CLL patients were incubated with 0.01-10 μM SAR409 for 48 hours. Apoptosis was analyzed by measuring DiOC6 signal in CD3+ T cells. Specific apoptosis was calculated (n=2).

Figure S9. BCR-controlled adhesion of mantle cell lymphoma cell line JeKo-1 to fibronectin is inhibited by SAR408. JeKo-1 cells pretreated with 1 µM SAR408 or SAR409 were stimulated with

α

IgM and allowed to adhere to fibronectin-coated surfaces (n=2). Graphs are presented as normalized mean ± SEM (100% = stimulated cells without inhibitors).

Supplemental Figure 8/Thijssen et al

T cells 0 0.01 0.1 1 10 0 20 40 60 80 100 SAR409 (µM) Sp eci fic ap op to si s ( % )

Supplemental Figure 9/Thijssen et al

contr ol contr ol SAR4 08 SAR4 09 0 50 100 αIgM R el at ive ad hesi on (% )