Supplementaryinformation

Table 1. Clinical characteristics of patients with B-CLL

All patients were untreated at the time of this study. The average age of the patients was 69 years in a group composed of 12 males and 16 females. The T cell-specific zeta-associated protein 70 (Zap 70) is an intracellular tyrosine kinase andserves to distinguish CLLsubtypes, even though it is not considered to be a reliable prognostic marker. The expression of ZAP-70 and the co-expression of the T-cell antigen CD5 and B-cell surface antigens CD19 were analyzed in peripheral-blood samples from the patients with CLL using specific antibodies and flow cytometry. Positive (over 15%) and negative (less than14%) signals are indicated by + and –, respectively, while ND indicates not determined. About 13% of the tested samples were ZAP-positive.

Identity / Age in years / Rai stage of disease / WBC
103/µl / Zap 70 / CD5/CD19
% / Gender
CLL 1 / 40 / 0 / 12 / ND / ND / M
CLL 2 / 73 / IV / 133 / ND / M
CLL 3 / 72 / 0-I / 39 / - / ND / F
CLL 4 / 75 / I / 19.7 / - / ND / F
CLL 5 / 78 / 0 / 12.2 / -/+ / ND / F
CLL 6 / 83 / I / 16.7 / ND / ND / F
CLL 7 / 58 / IV / 57.6 / + / ND / F
CLL 8 / 64 / II / 40 / + / ND / F
CLL 9 / 56 / III / 56.8 / - / ND / M
CLL 10 / 62 / I / 15.9 / - / ND / F
CLL 11 / 62 / 0 / 28.8 / + / 75.7 / F
CLL 12 / 74 / I / 15 / - / 32.7 / M
CLL 13 / 57 / I / 18.8 / - / ND / M
CLL 14 / 83 / I / 20 / ND / 60.7 / M
CLL 15 / 83 / I / 29 / - / 58.3 / F
CLL 16 / 75 / I / 27 / ND / 74 / M
CLL 17 / 70 / III / 43 / - / 64.8 / F
CLL 18 / 64 / 0-I / 20 / - / 64.7 / M
CLL 19 / 56 / I / 36 / - / 13.4 / F
CLL 20 / 71 / 0-I / 12 / ND / 68.3 / M
CLL 21 / 56 / I / 40 / - / 77.2 / M
CLL 22 / 63 / 0 / 17.8 / + / 53 / F
CLL 23 / 52 / III / 147 / - / 78 / F
CLL 24 / 68 / 0 / 18 / ND / 50.8 / M
CLL 25 / 84 / I / 67 / - / 83 / M
CLL 26 / 68 / 0-I / 13.6 / ND / 8.5 / M
CLL 27 / 80 / 0 / 33.4 / ND / 62 / F
CLL 28 / 59 / 0 / 13.4 / - / 46.9 / M
CLL 29 / 73 / I / 17 / ND / 60.6 / M
CLL 30 / 80 / II / 71 / ND / 50.4 / F
CLL 31 / 61 / 0 / 7 / - / 38.1 / M
CLL 32 / 75 / I / 31 / - / 67 / F
CLL 33 / 75 / II-III / 27.2 / ND / 81.6 / M
CLL 34 / 52 / III / 126 / -/+ / 95 / F
CLL 35 / 67 / IV / 58 / - / 78 / M
CLL 36 / 64 / IV / 64 / + / 90.1 / F
CLL 37 / 76 / 0 / 28 / + / 40.5 / F
CLL 38 / 80 / I / 23 / - / 74 / M
CLL 39 / 49 / I / 13 / - / 13 / F
CLL 40 / 72 / 0-I / 14.8 / ND / ND / M
CLL 41 / 75 / I / 15 / - / 13 / F
CLL 42 / 85 / I / 26 / ND / 81 / M
CLL 43 / 71 / II / 48 / - / 84 / F
CLL 44 / 64 / 0 / 15.7 / - / 18 / F
CLL 45 / 73 / II / 22.7 / - / ND / F
CLL 46 / 83 / I / 29.8 / - / 55 / F
CLL 47 / 78 / IV / 26.6 / ND / 65 / F
CLL 48 / 84 / 0 / 22 / - / 78 / F
CLL 49 / 92 / 0 / 9 / ND / ND / M
CLL 50 / 61 / IV / 40 / ND / ND / M
CLL 51 / 63 / IV / 64 / - / ND / M

Table 2. Summary of the results with various peptides based on LP4 or the N-terminal domain of VDAC1 inducing cell death of PBMCs derived from CLL patients and of MEC-1 cells.

PBMCs isolated from a CLL patient and MEC-1 cells (1 x106/ml) were incubated for 90 min with the indicated concentration of peptide and analyzed for cell death by PI staining and FACS analysis.Values represent the IC50mean ± SE, with the number of independent experiment presented (n).

IC50 , µM / Peptide sequence AA / Peptide / No
MEC-1 / CLL
2.5±0.1
(n=16) / 0.7±0.1
(n=10) / RQIKIWFQNRRMKWKK-LP4 43 / Antp-LP-4 / 1
1.6±0.3
(n=5) / 0.7±0.4
(n=3) / RQIKIWFQNRRMKWKK-LP4 43 / D-Antp-LP4 / 2
1.7±0.4
(n=7) / 0.3
(n=3) / KRRMKWKK-LP4 35 / MinAntp-LP4 / 3
1.4±0.1
(n=5) / 0.6±0.2
(n=3) / KRRMKWKK-LP4 35 / D-MinAntp-LP4 / 4
2±1.1
(n=2) / 1.1±0.2
(n=3) / Ac-KRRMKWKK-LP4 35 / Acetyl-D-MinAntp-LP4 / 5
>10
(n=3) / 2.8±1.4
(n=3) / RKKRRQRRRGG-LP4 38 / TAT-LP4 / 6
3.6±0.2
(n=18) / 1.7 ±0.2
(n=4) / HAIYPRH-LP4 34 / Tf-LP4 / 7
5±0.9
(n=3) / ND / HAIYPRH-LP4 34 / D-Tf-LP4 / 8
1.8±0.2
(n=3) / 1.2±0.1
(n=3) / HAIYPRH-LP4 34 / Tf-D-LP4 / 9
4.1±0.7
(n=3) / ND / Ac-HAIYPRH-LP4 34 / Acetyl-Tf-LP4 / 10
>6.8
n=3)) / ND / HAIYPRH-LP4-NH2 34 / Tf-LP4-amid / 11
>15
n=2)) / ND / HAIYPRH-Linear 24 / Tf-Linear / 12
8.8
n=2)) / ND / HAIYPRH-Cyclic 24 / Tf-Cyclic / 13
3.5±0.4
(n=3) / 3.5±0.7
(n=3) / RQIKIWFQNRRMKWKK-NL 25 / Antp-No-Loop / 14
>15
(n=3) / ND / KRRMKWKK-NL 17 / MinAntp-No-Loop / 15
>14.3
(n=3) / >10.7
(n=3) / HAIYPRH-NL 16 / Tf-No-Loop / 16
4.23±0.2
n=12)) / 3.2±0.2
n=6)) / N-Ter-RQIKIWFQNRRMKWKK 42 / N-Ter-Antp / 1111 17
>20
n=3)) / >30
(n=3) / N-Ter-RKKRRQRRRGG 37 / N-Ter-TAT / 18
>16.7
(n=3) / >25
(n=3) / N-Ter-HAIYPRH 33 / N-Ter-Tf / 19
2.2±1.6
(n=3) / 1.5±0.6
(n=3) / N-Ter-RQIKIWFQNRRMKWKK 42 / D-N-Ter-Antp / 20
>12.5
(n=3) / >12.5
(n=3) / ∆(21-26)N-Ter-RQIKIWFQNRRMKWKK36 / N-Ter∆21-26-Antp / 21
4.2±0.2
(n=4) / ND / ∆(1-4)N-Ter-RQIKIWFQNRRMKWKK 38 / ∆1-4N-Ter-Antp / 22
2.5±0.2 (n=7) / ND / ∆(1-10)N-Ter-RQIKIWFQNRRMKWKK 32 / ∆1-10N-Ter-Antp / 23
1.9±0.2
(n=3) / ND / ∆(1-10)N-Ter-RQIKIWFQNRRMKWKK 32 / D-∆1-10N-Ter-Antp / 24
3.3±0.2 (n=7) / 2.2±0.2
(n=3) / ∆(1-14)N-Ter-RQIKIWFQNRRMKWKK 28 / ∆1-14N-Ter-Antp / 25
2.1±0.3 (n=3) / 1.3±0.1
(n=2) / ∆(1-14)N-Ter-RQIKIWFQNRRMKWKK 28 / D-∆1-14N-Ter-Antp / 26
>15
(n=4) / ND / ∆(1-10)N-Ter-RRMKWKK 24 / ∆1-10N-Ter-MinAntp / 27

The sequences of the abbreviations used are:

LP4: SWTWEKKLETAVNLAWTAGNSNKWTWK

N-Ter: MAVPPTYADLGKSARDVFTKGYGFGL

NL(No-loop): LETAVNLAW

Linear: KKLETAVNLAWTAGNSN

Cyclic: CKKLETAVNLAWTAGNSNC

Ac=Acetyl, NH2 = amid, AA= number of amino acids

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