The interplay between microenvironmental signaling and novel targeted drugs in CLL - Chapter 5: Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20

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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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RESISTANCE TO ABT-199 INDUCED BY

MICROENVIRONMENTAL SIGNALS IN

CHRONIC LYMPHOCYTIC LEUKEMIA CAN BE

COUNTERACTED BY CD20 ANTIBODIES OR

KINASE INHIBITORS

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Rachel Thijssen1,2_{, Erik Slinger}1,2_{, Katinka Weller}1_{, Christian R. Geest}1_{, Tim Beaumont}3_,

Marinus H.J. van Oers2,4_{, Arnon P. Kater}2,4,5_{, Eric Eldering}1,4,5

Departments of Experimental Immunology1_{and Hematology}2_{, Academic Medical Center, University of} Amsterdam, Amsterdam, The Netherlands

3_{AIMM Therapeutics, Amsterdam, the Netherlands}

4_{Lymphoma and Myeloma Center Amsterdam, LYMMCARE}

5_{Shared senior authorship}

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YING SENSITIVITY TO ABT

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A major clinical problem in chronic lymphocytic leukemia (CLL) is (development of) chemoresistance, which can be caused by genetic lesions but is also strongly influenced by the leukemic microenvironment. Two compartments can be distinguished in CLL; the blood, where quiescent CLL cells accumulate, and the lymphoid ‘microenvironment’ within the lymph nodes (LN), spleen and bone marrow, where surrounding cells provide external signals that drive CLL proliferation and survival1_{. CLL survival is correlated with}

NF-

κ

B-mediated upregulation of various protective Bcl-2 family members, notably Bcl-XL, Bfl-1 and Mcl-1 in LN samples compared to peripheral blood (PB)2;3_.

Recently, novel therapeutics that target microenvironmental signals or Bcl-2 family members have entered clinical trials and practice1;4_{. One prominent strategy for CLL and}

other cancers is to target the apoptosis machinery directly by so-called BH3-mimetics. The Bcl-2-specific compound ABT-199 or Venetoclax is highly cytotoxic for CLL cells and shows improved clinical efficacy and induces no trombocytopenia as opposed to its predecessor Navitoclax4_{. Peripheral blood lymphocyte counts, lymph node size and bone}

marrow involvement all diminished early after treatment5_{. A second strategy employs}

kinase inhibitors that target critical signal transduction pathways controlling cell growth, adhesion and survival. Inhibitors of the B cell receptor (BCR)-associated kinases Bruton’s tyrosine kinase (Btk) and phosphatidylinositol-3-kinase (PI3K) show strong clinical activity and were recently approved for both relapsed/refractory CLL and for CLL harboring 17p deletions6;7_{. A third novel strategy is treatment with next-generation anti-CD20 monoclonal}

antibodies (mAb), e.g. with enhanced direct cytotoxicity and antibody-dependent cell mediated cytotoxicity (GA101, obinutuzumab)8_.

As outlined above, pro-survival signals can upregulate Bcl-2 members which are not targeted by ABT-199. We therefore analyzed to what extent microenvironmental signals can affect sensitivity to ABT-199 in CLL samples, and whether this can be counteracted by combination therapy with other novel therapeutics. This study was approved by the AMC Ethical Review Board and conducted in agreement with the Declaration of Helsinki (see Supplemental Table 1 for patient characteristics).

We first established by immunofluorescence that Bcl-XL is expressed in LN CLL cells. For comparison, we performed immunostaining on CLL cells stimulated with CD40 ligand in vitro, mimicking activated T cell-mediated signalling9_{. This also showed}

clear Bcl-XL expression, whereas PB CLL cells were essentially negative (Figure 1, Supplemental Figure 1). We next applied in vitro CD40 stimulation with IL-21 and IL-4, cytokines produced by activated T cells and follicular helper T cells that can induce proliferation and survival, respectively9;10_{. As reported before}10;11_{, CD40 stimulation of}

CLL cells increased expression of Bcl-2 members Mcl-1, Bcl-XL and Bfl-1 (Figure 2A). T cell cytokines had contrasting effects; IL-21 downregulated Bcl-XL while IL-4 further increased Bcl-XL and Bfl-1 expression. Mcl-1 was not significantly altered by addition of IL-21 or IL-4 (see Supplemental Figure 2 for averaged quantification on multiple samples). Unstimulated CLL cells are highly sensitive to ABT-199 (LC50 < 1nM, see Supplemental Table 2). Remarkably, CD40 and CD40+IL-4 stimulation resulted in full resistance to 10 μM ABT-199. The effect of IL-21 was less pronounced, probably as a result of downregulated Bcl-XL in comparison with CD40 stimulation alone (Figure 2A,B). There were differences among patient samples in the basal sensitivity and extent of resistance

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5 Figure 1

B

A

DAPI Mouse IgG Rabbit IgG DAPI CD20 Bcl-XL DAPI CD20 Bcl-XL

C

D

DAPI CD20 Bcl-XL

PBMC – 3T3

PBMC – 3T40L

Lymphnode

Figure 1. Bcl-XL is expressed in CLL cells in LN tissue. One representative CLL LN sample out of 4 stained is shown and stained for A) Isotype control staining and nuclear staining with DAPI in blue, scale-bar represent 10 µm. B) CD20 in green and Bcl-XL in red and nuclear staining with DAPI in blue C-D). CLL cells (Supplemental table 1; patient #23-25) were co-cultured with NIH3T3 fibroblasts transfected with empty vector (3T3) (C) or co-cultured with NIH3T3 fibroblasts transfected with hCD40L (3T40L) (D) for 3 days. After detachment, cytospins were made and stained for CD20, Bcl-XL and DAPI. Imaging was performed using a Leica TCS SP8-X confocal microscope. One CLL sample is shown of a total of three analyzed, scale-bar represent 10 µm. A-D) Paraffin-embedded LN samples from CLL patients were incubated with primary antibody anti-CD20 (eBioscience, San Diego, CA, USA) and anti-Bcl-XL (Cell Signaling, Boston, MA, USA) and subsequently incubated with Alexa Fluor 488 labeled goat anti-mouse and Alexa Fluor 594 labeled goat anti-rabbit antibodies (Invitrogen, Camarillo, CA, USA) and counterstained with DAPI. Immunofluorescent imaging (40x) was performed using a Leica DMRA fluorescence microscope.

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Figure 2

B

A

0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 ABT-199 (µM) Via bil ity (% D iO C 6+ ) 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 _3T3 3T40L 3T40L + IL-21 3T40L + IL-4 ABT-199 (µM) Spec ifi c apopt os is (% )

C

0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 _3T3 3T40L+siCtrl 3T40L+siBclX 3T40L+siMcl1 ABT-199 (µM) Spec ifi c apopt os is (% ) 3T3 3T40L siCtrl siBclX siMcl1

Mcl-1 Bcl-XL Actin Mcl-1 Bcl-XL Actin 3T3 3T40L +IL-21 +IL-4 Patient #10B Patient #11 Bfl-1/A1 Bcl-2 3T3 3T40L +IL-21 +IL-4

Figure 2. CLL cells become resistant to ABT-199 upon CD40 stimulation. CLL cells were cultured on control 3T3 or 3T40L cells in the presence or absence of 25 ng/ml IL-21 or IL-4 for 72 hours. A) Western blot analysis was performed using standard techniques with anti-Mcl-1, 2, anti-Bcl-XL, anti-Bfl-1 and anti-

β

-actin antibodies. Blots from two representative CLL samples are shown of a total of seven analyzed (Supplemental table 1; patient #1-3A, 10B, 11). B) After detachment, cells were incubated with 0.001-10 μM ABT-199 for 24 hours. Viability was assessed by DiOC6/PI staining. Left panel shows % viable cells, right panel shows specific apoptosis. Results are shown as mean ± SEM, n=8 (3 IgVH mutated, 3 unmutated, 2 IgVH status unknown (Supplemental table 1; patient #4-11) C) CLL cells (Supplemental table 1; patient #5, 13A, 19) were nucleofected (Amaxa, Koln, Germany) with 3 µg siRNA (Bcl-XL, Mcl-1 and Silencer Select Negative Control) and cultured on 3T40L cells for 3 days. Left panel: lysates were probed for Bcl-XL, Mcl-1 or actin as a loading control. As a control lysates of CLL cells on 3T3 are shown. Right panel, after nucleofection and culture, cells were incubated with ABT-199 for 24 hours, n=3, mean ± SEM. Reagents: anti-Mcl-1 (Cell Signaling), Bcl-2 (Enzo Life Sciences, Raamsdonksveer, The Netherlands), Bcl-XL (BD Biosciences),

anti-β

-actin (Santa Cruz Biotechnology), polyclonal antibody against Bfl-1 was a kind gift of Jannie Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands), IL-21 and IL-4 (Gibco, Invitrogen, Bleiswijk, The Netherlands), ABT-199 from Abbvie (Abbott Park, IL, USA), siRNA from obtained from Ambion (ID#1920, ID#8583 and Cat#4390843). Specific apoptosis is defined as [% cell death in treated cells] – [% cell death in medium control] / [% viable cells medium control] x 100.

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induction towards BH3-mimetics, which were reproducible in independent experiments (Supplemental Figure 3). No differences in response were observed among mutated (n=4) versus unmutated (n=4) IgV_H CLL samples. Nucleofection with Bcl-XL siRNA resulted in restored susceptibility for ABT-199. Unexpectedly, knock-down of Mcl-1 hardly altered the sensitivity to ABT-199 after CD40 stimulation (Figure 2C), although it slightly decreased spontaneous apoptosis (data not shown). Unfortunately, no clear knock down of Bfl-1 could be obtained for unknown reasons. Thus, in the context of CD40 stimulation, especially Bcl-XL plays an important role in shifting the susceptibility of CLL cells for ABT-199. The role of individual Bcl-2 members was separately addressed in immortalized primary B cells as described before11_{, which established that both Bfl-1 and Mcl-1 can also singly}

confer resistance to ABT-199 when expressed at sufficient levels (Supplemental Figure 4). Next, we investigated combination strategies to revert resistance for ABT-199 induced by CD40 stimulation. Previously we have demonstrated that, whereas unstimulated CLL cells are not sensitive to CD20-mediated killing, anti-CD20 monoclonal antibodies induce non-apoptotic cell death in CD40-stimulated CLL cells12;13_{. Combination of GA101 or}

rituximab with ABT-199 demonstrated that CD40-induced resistance to ABT-199 could be counteracted by anti-CD20 antibodies (Figure 3A). As shown elsewhere, treatment with anti-CD20 Abs does not affect expression of Bcl-2 family members in CD40-stimulated CLL cells12_.

Secondly, in agreement with previous studies using ABT-73711_{, resistance to ABT-199 in}

CLL could also be significantly reverted by the Src kinase inhibitor dasatinib at physiologically relevant doses (Figure 3B). Prime targets of dasatinib are Abl and Btk14_{. The Btk inhibitor}

ibrutinib but also the PI3K

δ

inhibitor idelalisib have not been investigated in the context of CD40-mediated drugresistance. We found that ibrutinib and idelalisib had minimal impact on CD40-induced resistance for ABT-199 (Figure 3C). It should be noted in this context that additional BCR stimulation had no effect on CD40-induced resistance for ABT-199 or on induction NF-

κ

B responsive Bcl-2 family members (Supplemental Figure 5). In contrast, high dose of the c-Abl inhibitor imatinib could reverse the CD40-induced resistance for ABT-199. In agreement with their effects on ABT-199 sensitivity, c-Abl inhibitors dasatinib and imatinib reversed induction of pro-survival Bcl-XL, Mcl-1, and Bfl-1, whereas ibrutinib or idelalisib had no effect (Figure 3D). Of note, the kinase inhibitors by themselves hardly induced apoptosis upon CD40 stimulation of CLL cells (Supplemental Figure 6). Supplemental table 2 summarizes the effects of the various stimulations and combination therapies on the LC₅₀ for ABT-199.

Development or selection of resistant clones is a major problem in cancer treatment. Both for conventional chemotherapeutics and novel targeted drugs, there is considerable selection pressure for the cancer cells to escape elimination. Our in vitro co-culture model of CLL applies prolonged CD40 stimulation, and represents an extreme system of chemoprotection, where primary CLL cells are converted within three days from highly sensitive to fully resistant to ABT-199. More moderate forms of pro-survival signalling will occur in vivo and are also observed by co-culture of CLL cells with autologous activated T cells9_{, and this is also supported by the modulating effect of IL-21 in vitro. Still, in CLL}

lymph nodes upregulation of Bcl-XL, Bfl-1 and Mcl-1 can be observed2;3_{, and this might be}

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Figure 3

A

B

0 0,001 0,1 10 0 20 40 60 80 100 3T3 3T40L 3T40L + GA101 3T40L + RXL * ** * *** *** *** *** ABT-199 (µM) % cel l d eat h ( D iO C 6-) 0 0.001 0.1 10 0 20 40 60 80 100 3T3 3T40L 3T40L + 0.1 µM dasatinib 3T40L + 1 µM dasatinib * * *** *** *** *** ABT-199 (µM) % cel l d eat h ( D iO C 6-)

C

D

Mcl-1 Bfl-1 das ima ibr inhibitors 3T40L - 0 0.001 0.1 10 0 20 40 60 80 100 3T3 3T40 3T40 + 1 µM Ibrutinib 3T40 + 30 µM Imatinib 3T40 + 1 µM Idelalisib *** *** *** *** ABT-199 (µM) % cel l d eat h ( D iO C 6-) Bcl-XL actin 3T40L ide 3T3 3T3

Figure 3. CD40L-induced resistance to ABT-199 can be overcome by combination therapy. A) CLL cells were co-cultured with 3T3 or 3T40L for 3 days with 10 µg/ml crosslinked Rituximab (RXL) or GA101 as described12;13_{, or in combination with indicated concentrations of ABT-199 and analysed} for apoptosis after 24 hours. Averaged results are presented as percentage cell death (mean ± SEM) for 6 CLL samples (Supplemental table; patient #3B, 5, 7, 10B, 19, 22) for GA101 and 3 CLL samples (Supplemental table; patient #3B, 7, 10B) for Rituximab. B) CLL cells were co-cultured in the presence of 0.1 or 1 μM dasatinib and after deatchment were incubated with ABT-199. (n=8 Supplemental table; patient #5, 8, 10B-12, 19-21), C) CLL cells were cocultured in the presence of 1 μM ibrutinib, 1 μM idelalisib or 30 μM imatinib. After detachment, cells were incubated with ABT-199. (n=8 Supplemental table; patient #3B-5, 8, 12, 13B-15). D) CD40-stimulated CLL cells were co- cultured for 48 hours in the presence of dasatinib, imatinib, or ibrutinib as indicated. Lysates were probed for Mcl-1, Bcl-XL and Bfl-1 and actin for loading control. The results are representative

of 3 blots. Reagents: GA101 (Roche, Woerden, The Netherlands) , dasatinib (Novartis,

Basel, Switzerland), ibrutinib (Pharmacyclics, Sunnyvale, CA, USA), idelalisib (Selleckchem, Houston, TX, USA), imatinib (Novartis, Basel, Switzerland). Statistics by Students T-test: * p <0.05;** p<0.01; *** p<0.001.

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Support for this possibility has been obtained in vitro by selection of clones resistant to ABT-199, caused by mutation in Bcl-2 or Bax15_{. To obtain long-term and complete CLL}

remission in vivo, combination strategies of ABT-199 with kinase inhibitors or anti-CD20 antibodies might be attractive. The clinical activities of dasatinib, ibrutinib or idelalisib can be attributed to inhibition of BCR-mediated survival and adhesion, followed by egress of CLL cells from LN into the bloodstream. There, they express almost exclusively Bcl-2 and are therefore highly susceptible to ABT-199. The combination of ABT-199 with anti-CD20 monoclonal antibodies is currently being explored in clinical trials. With regard to direct cell death induction, our findings support the effectiveness of such combinations in CLL in relation to drug-resistant niches at lymph-node sites.

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.

CONFLICT OF INTEREST STATEMENTS

The authors declare no competing financial interests.

AUTHORS CONTRIBUTION

RT performed experiments, interpreted data and wrote the paper, ES performed immunofluorescence, KW and CRG performed and interpreted experiments, TB supervised experiments with immortalized primary B cells, MHJvO provided patient samples and edited the paper, APK suggested and interpreted experiments and edited the paper, EE supervised the study and wrote the paper.

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REFERENCES

1. ten Hacken E., Burger JA. Molecular pathways: targeting the microenvironment in chronic lymphocytic leukemia--focus on the B-cell receptor. Clin Cancer Res. 2014;(20):548-556. 2. Smit LA, Hallaert DY, Spijker R et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node

chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668. 3. 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):5071-5082.

4. Souers AJ, Leverson JD, Boghaert ER et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat.Med. 2013;(19):202-208.

5. Seymour JF, Davids MS, Pagel JM et al. Updated results of a phase I first-in-human study of the Bcl-2 inhibitor ABT-199 (GDC-0199) in patients with relapsed/refractory R/R chronic lymphocytic leukemia (CLL) [abstract]. ASCO 2013

6. 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):3390-3397. 7. Byrd JC, Furman RR, Coutre SE et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic

leukemia. N.Engl.J.Med. 2013;(369):32-42.

8. Mossner E, Brunker P, Moser S et al. Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood 2010;(115):4393-4402.

9. 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):3010-3019.

10. Vogler M, Butterworth M, Majid A et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia.

Blood 2009;(113):4403-4413.

11. 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):487-498.

12. Jak M, van Bochove GG, van Lier RA, Eldering E, van Oers MH. CD40 stimulation sensitizes CLL cells to rituximab-induced cell death. Leukemia 2011;(25):968-978.

13. Jak M, van Bochove GG, Reits EA et al. CD40 stimulation sensitizes CLL cells to lysosomal cell death induction by type II anti-CD20 mAb GA101. Blood 2011;(118):5178-5188.

14. Hantschel O, Rix U, Schmidt U et al. The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc.Natl.Acad.Sci.U.S.A 2007;(104):13283-13288.

15. Fresquet V, Rieger M, Carolis C, Garcia-Barchino MJ, Martinez-Climent JA. Acquired mutations in BCL2 family proteins conferring resistance to the BH3 mimetic ABT-199 in lymphoma.

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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 WBC 10^9/L % lymphocytes %CD19+/CD5+ cells FISH Tr eatment befor

e sampling mutational status

1 79 M U 122, 0 n. d. 82, 0 tri s 1 2 chl or am buc il 2 62 M U 155, 0 96, 0 79, 1 tr is 12, 17p-chl or am buc il, pr edni sone, dex am et has one 3A 72 F U 115, 3 n. d. 90, 3 tri s 1 2 none 3B 73 F U 89, 9 n. d. 91, 4 tri s 1 2 none 4 68 F U 100, 5 89, 0 84, 8 tri s 1 2 none 5 60 F U 265, 0 n. d. 99, 8 13q-FCR 6 74 M n. d. 58, 4 86 92, 7 n. d. none 7 66 M M 93, 4 n. d. 90, 8 none none 8 68 F M 65, 1 91, 4 78, 4 13q-chl or am buc il+ pr edni sone, s ter oi ds 9 69 F n. d. 27, 8 n. d. 81, 3 n. d. chl or am buc il 10A 73 M U 116, 8 n. d. 98, 4 n. d. none 10B 73 M U 166, 6 n. d. 84, 2 n. d. chl or am buc il 11 67 F M 88, 7 n. d. 86, 9 13 q - I g H/ CCND1 chl or am buc il, fl udar abi ne, pr edni sone 12 72 M U 108, 0 94, 7 96, 4 17p-chl or am buc il, fl udar abi ne, A lem tuz um ab, F C R 13A 62 F M 173, 0 n. d. 99, 9 q-, 17p-, I gH /C C N D none 13B 63 F M 153, 0 96, 0 95, 7 q-, 17p-, I gH /C C N D chl or am buc il, F C R 14 80 M M 149, 4 92, 0 98, 2 11q-, 13q-chl or am buc il 15 59 M M 85, 5 94, 0 95, 8 n. d. none 16 61 F M 46, 1 n. d. 91, 1 13q-none 17 59 M U 74, 0 n. d. 92, 3 11q-O fat um um ab 18 66 M U 232, 0 96, 1 98, 5 n. d. unk now n 19 63 F M 79, 2 91, 0 92, 3 n. d. none 20 55 F U 272, 0 n. d. 95, 4 none none 21 76 F M 112, 0 n. d. 91, 8 n. d. none 22 57 F M 170, 9 n. d. 97, 3 tr is 12, 13q-chor am buc il, fl udar abi ne 23 64 F U 64, 8 82 88 n. d. none 24 74 F n. d. 55, 0 n. d. 86, 4 n. d. none 25 60 M n. d. n. d. n. d. 97, 2 n. d. none

n.s . not s peci fi ed; n.d. not deter

mi ned; A, B, C r efer to di f fer ent s a mpl i ng ti mes of the s a

me pa ti ent; FCR: fl uda ra bi ne + cycl ophos pha mi de + ri tuxi ma b

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Patient 1

CD20 Bcl-XL

Patient 2

CD20 Bcl-XL

Patient 3

CD20 Bcl-XL

Patient 4

CD20 Bcl-XL

Supplemental Figure 1. Bcl-XL staining in lymph nodes from different CLL patients.

Immunofluorescent staining for CD20 (green) and Bcl-XL (red) showing presence of

Bcl-XL in CD20-positive cells in the lymph node of 4 CLL patients. Additional staining

for Patient 1 is also shown in Figure 1 of the main article.

Supplemental Figure 1

Table S2. LC50 values for ABT-199 and ABT-737 for CLL cells under various conditions. LC50 values were calculated from averaged data from CLL samples tested for sensitivity for ABT-199 or ABT-737 under the indicated conditions, n=8, n=3 for GA-101. (n.d. not determined)

Stimulation LC50 (μM) ABT-199 ABT-737 3T3 (Control) 0.001 0.005 3T40L >10 0.781 3T40L + 0.1 μM dasatinib 0.066 0.081 3T40L + 1 μM dasatinib 0.020 0.037 3T40L + 10 ug/ml GA-101 0.044 n.d. 3T40L + 10 ug/ml RXL 0.065 n.d.

Figure S1. Bcl-XL staining in lymph nodes from different CLL patients. Immunofluorescent staining for CD20 (green) and Bcl-XL (red) showing presence of Bcl-XL in CD20-positive cells in the lymph node of 4 CLL patients. Additional staining for Patient 1 is also shown in Figure 1 of the main article.

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5 Supplemental Figure 2

3T3 3T40 L 3T40 L + IL -21 3T40 L + IL -4 0 2 4 6 8 Mc l-1 /β -a ct in 3T3 3T40 L 3T40 L + IL -21 3T40 L + IL -4 0 2 4 6 8 ** * * Bc l-XL /β -a ct in 3T3 3T40 L 3T40 L + IL -21 3T40 L + IL -4 0 5 10 15 B fl-1 /β -a ct in *

Supplemental Figure 2. Stimulation via CD40 plus IL-4 or IL-21 differentially induces

expression of Mcl-1, Bcl-XL and Bfl-1/A1. Densitometric analysis of Mcl-1/actin

levels, Bcl-XL/actin levels and Bfl-1/actin levels of seven CLL samples is shown. Bars

represent the mean ± SEM, * p <0.05;** p<0.01. Western blots from 2 patients are

shown in Figure 2A of the main article.

Figure S2. Stimulation via CD40 plus IL-4 or IL-21 differentially induces expression of Mcl-1, Bcl-XL and Bfl-1/A1. Densitometric analysis of Mcl-1/actin levels, Bcl-XL/actin levels and Bfl-1/actin levels of seven CLL samples is shown. Bars represent the mean ± SEM, * p <0.05;** p<0.01. Western blots from 2 patients are shown in Figure 2A of the main article.

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Supplemental Figure 3

pt #10B 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 3T3-1 3T3-2 3T40L-2 3T40L-1 ABT-199 (µM) Sp eci fic ap op to si s ( % ) pt #10B 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 3T3-1 3T3-2 3T40L-1 3T40L-2 ABT-737 (µM) Sp eci fic ap op to si s ( % ) pt #8 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 3T3-1 3T3-2 3T40L-1 3T40L-2 3T40L-3 3T3-3 ABT-199 (µM) Sp eci fic ap op to si s ( % ) pt #8 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 3T3-1 3T40L-1 3T3-2 3T40L-2 3T3-3 3T40L-3 ABT-737 (µM) Sp eci fic ap op to si s ( % ) pt #5 0 0,001 0 20 40 60 80 100 3T3-2 3T40L-2 3T3-1 3T3-3 3T40L-1 3T40L-3 ABT-199 (µM) Sp eci fic ap op to si s ( % ) pt #5 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 3T3-2 3T40L-2 3T3-3 3T40L-3 3T3-1 3T40L-1 ABT-737 (µM) Sp eci fic ap op to si s ( % ) pt #4 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 _3T3-1 3T3-2 3T40L-1 3T40L-2 ABT-199 (µM) Sp eci fic ap op to si s ( % ) pt #4 0 0,001 0,01 0,1 1 10 0 20 40 60 80 100 _3T3-1 3T3-2 3T40L-1 3T40L-2 ABT-737 (µM) Sp eci fic ap op to si s ( % )

A

B

Supplemental Figure 3. Heterogeneity among patients in the response to BH3-mimetics after CD40 stimulation is reproducible. A-B. CLL cells were stimulated with 3T3 or 3T40L cells for 3 days. After

detachment, cells were incubated with ABT-199 (A) or ABT-737 (B) for 24 hours. Specific apoptosis of two or three independent experiments are shown for 4 patient samples.

Figure S3. Heterogeneity among patients in the response to BH3-mimetics after CD40 stimulation is reproducible. A-B. CLL cells were stimulated with 3T3 or 3T40L cells for 3 days. After detachment, cells were incubated with ABT-199 (A) or ABT-737 (B) for 24 hours. Specific apoptosis of two or three independent experiments are shown for 4 patient samples. Figure S4. Immortalized primary B cells with overexpression of Mcl-1, Bfl-1, Bcl-XL or knockdown of Noxa are resistant to ABT-199. A. Primary human memory B cells were immortalized by overexpressing Bfl-1/A1, Bcl-2, Bcl-XL, Mcl-1 or knockdown of Noxa (described in Tromp et al, Clin Cancer res 2012, 18: 487). Overexpression or knockdown was confirmed by Western blot analysis. B. Immortalized primary B cells with overexpression of Bcl-2 or Bcl-XL were incubated with different concentration of ABT-737 or ABT-199. Bars represent the mean specific apoptosis ± SEM, n=3. C. Immortalized primary B cells with overexpression of Bfl-1/ A1, Mcl-1 or knockdown of Noxa were incubated with different concentration of ABT-737 or ABT-199.

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5 A

B

0.0 2.5 5.0 7.5 10.0 0 20 40 60 80 100 _{Bfl-1/A1 ABT-737} Mcl-1 ABT-737 Noxa knock down ABT-737 Bfl-1/A1 ABT-199 Mcl-1 ABT-199 Noxa knock down ABT-199

Concentration (µM) Sp eci fic ap op to si s ( % ) 0.0 2.5 5.0 7.5 10.0 0 20 40 60 80 100 ABT-737 ABT-199 Bcl-2 Concentration ( µM) Sp eci fic ap op to si s (% ) 0.0 2.5 5.0 7.5 10.0 0 20 40 60 80 100 ABT-737 ABT-199 Bcl-XL Concentration ( µM) Sp eci fic ap op to si s ( % )

C

Supplemental Figure 4

Supplemental Figure 4. Immortalized primary B cells with overexpression of Mcl-1, Bfl-1, Bcl-XL or

knockdown of Noxa are resistant to ABT-199. A. Primary human memory B cells were

immortalized by overexpressing Bfl-1/A1, Bcl-2, Bcl-XL, Mcl-1 or knockdown of Noxa (described in

Tromp et al, Clin Cancer res 2012, 18: 487). Overexpression or knockdown was confirmed by

Western blot analysis. B. Immortalized primary B cells with overexpression of Bcl-2 or Bcl-XL were

incubated with different concentration of ABT-737 or ABT-199. Bars represent the mean specific

apoptosis ± SEM, n=3. C. Immortalized primary B cells with overexpression of Bfl-1/A1, Mcl-1 or

knockdown of Noxa were incubated with different concentration of ABT-737 or ABT-199.

Figure S4. Immortalized primary B cells with overexpression of Mcl-1, Bfl-1, Bcl-XL or knockdown of Noxa are resistant to ABT-199. A. Primary human memory B cells were immortalized by overexpressing Bfl-1/A1, Bcl-2, Bcl-XL, Mcl-1 or knockdown of Noxa (described in Tromp et al, Clin Cancer res 2012, 18: 487). Overexpression or knockdown was confirmed by Western blot analysis. B. Immortalized primary B cells with overexpression of Bcl-2 or Bcl-XL were incubated with different concentration of ABT-737 or ABT-199. Bars represent the mean specific apoptosis ± SEM, n=3. C. Immortalized primary B cells with overexpression of Bfl-1/A1, Mcl-1 or knockdown of Noxa were incubated with different concentration of ABT-737 or ABT-199.

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Supplemental Figure 5

0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 control αIgM CD40L CD40L + αIgM ABT-199 (µM) Sp eci fic ap op to si s ( % )

C

CD40L

αIgM αIgM

C

CD40L

αIgM αIgM

C

CD40L

αIgM αIgM

Patient #2

Patient #20

Patient #23

Bcl-XL

Bfl-1

actin

A

B

Supplemental Figure 5. Effect of combined CD40+ BCR stimulation on sensitivity for ABT-199, and expression of Bcl-2 family members. CLL cells were cultured with medium or 3T40L in the presence

or absence of 500ng/ml goat (Fab’)2 anti-human IgM (Sanbio, Uden, The Netherlands) for 3 days.

A. After detachment, cells were incubated with 0.001-10 μM ABT-199. Results are shown as mean ±

SEM, n=3 (IgVH unmutated) (Supplemental table 1; patient #2, 20, 23). B. protein lysates were

probed for Bcl-XL, Bfl-1 and actin as loading control.

Figure S5. Effect of combined CD40+ BCR stimulation on sensitivity for ABT-199, and expression of Bcl-2 family members. CLL cells were cultured with medium or 3T40L in the presence or absence of 500ng/ml goat (Fab’)2 anti-human IgM (Sanbio, Uden, The Netherlands) for 3 days. A. After detachment, cells were incubated with 0.001-10 μM ABT-199. Results are shown as mean ± SEM, n=3 (IgVH unmutated) (Supplemental table 1; patient #2, 20, 23). B. protein lysates were probed for Bcl-XL, Bfl-1 and actin as loading control.

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Figure S6. Direct killing effects of various kinase inhibitors before and after CD40 stimulation. A-B) CLL cells were thawed and incubated with dasatinib, imatinib and ibrutinib for 48 hours. Results are shown as mean ± SEM, n=3 for IgVH mutated CLL cells (A) and n=3 for IgVH unmutated CLL cells (B). C) CLL cells were stimulated with 3T3 or 3T40L cells for 3 days. After detachment, cells were incubated with dasatinib for 48 hours. Viability of 4 CLL samples are shown. D) CLL cells were cocultured with 3T3 or 3T40L for 72 hours, in the presence of imatinib, ibrutinib or idelalsib as indicated. After detachment, viability was assessed and averaged data of 8 CLL samples are shown, error bars represent SEM.

Supplemental Figure 6

3T3 mediu m M Ibr utinib µ 1 M Idelal isib µ 1 M Imati nib µ 1 M Imati nib µ 30 0 20 40 60 80 100 3T40L Via bilit y ( % D iO C 6+ ) 0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 pt #5 3T3 pt #13B 3T3 pt #23 3T3 pt #7 3T3 pt #5 3T40L pt #13B 3T40L pt #23 3T40L pt #7 3T40L Dasatinib (µM) Via bilit y ( % D iO C 6+ )

IgVH mutated CLL cells

0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 Ibrutinib Imatinib Dasatinib Concentration (µM) Via bilit y ( % D iO C 6+ )

IgVH unmutated CLL cells

0 0.001 0.01 0.1 1 10 0 20 40 60 80 100 Ibrutinib Imatinib Dasatinib Concentration (µM) Via bilit y ( % D iO C 6+ )

A

B

C

D

Supplemental Figure 6. Direct killing effects of various kinase inhibitors before and after CD40 stimulation. A-B) CLL cells were thawed and incubated with dasatinib, imatinib and ibrutinib for 48

hours. Results are shown as mean ± SEM, n=3 for IgVH mutated CLL cells (A) and n=3 for IgVH unmutated CLL cells (B). C) CLL cells were stimulated with 3T3 or 3T40L cells for 3 days. After detachment, cells were incubated with dasatinib for 48 hours. Viability of 4 CLL samples are shown.

D) CLL cells were cocultured with 3T3 or 3T40L for 72 hours, in the presence of imatinib, ibrutinib or

idelalsib as indicated. After detachment, viability was assessed and averaged data of 8 CLL samples are shown, error bars represent SEM.