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patients with a variety of B-cell malignancies. While p17AE2 showed a moderate effect on normal B lympho- cytes, decreasing B-cell viability only at high dosage (50 μg/mL) (Figure 3A), this peptide markedly reduced cell growth in 44 of 69 (64%) malignant cell lines, as fol- lows: 28 of 43 (65%) mature B-cell lymphomas (8 of 12 mantle cell lymphoma, 5 of 8 Burkitt lymphoma, 14 of 21 diffuse large B-cell lymphoma and 1 of 2 splenic mar- ginal-zone B-cell lymphoma); 2 of 4 EBV transformed lymphoblastoid B-cell lines; 9 of 16 (56%) multiple myelomas; and 5 of 6 (83%) B-cell acute lymphoblastic leukemias (Figure 3B). The effects of p17AE2 on cell via- bility and apoptosis were dose-dependent in p17AE2 sensitive B-cell lymphoma cell lines, but not in resistant OCI-Ly1 B-cell lymphoma cells (Figure 3 C-D). Cell cycle, however, was not affected either in sensitive or in resistant cell lines (Figure 3E). Besides, p17AE2 also affected cell viability and apoptosis of several acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL) (Figure 3 F-G). These results indicate that targeting AE2 with p17AE2 reduces cell survival in a variety of hematologic neoplasms, including B-cell tumors, while showing only moderate effects on non- tumoral B lymphocytes.
Functional targeting peptide p17AE2 induces apoptosis in tumor cells by modulating intracellular pH and AE2 function
To study how p17AE2 reduces cell viability of tumor B cells after targeting AE2 protein, basal pHi values were measured in normal B cells and in tumor B-cell lines. Consistent with previous studies, human peripheral blood B lymphocytes from healthy donors showed basal pHi values ranging from 7.0 to 7.2, whereas cell lines derived from different B-cell malignancies exhibited higher pH
pH is permissive for tumor cell proliferation and evasion i
of apoptosis, while acidic pH values promote cell apopto- i
sis.10,14 Incubation of tumor B cells with p17AE2 led to dose-dependent pHi acidification in sensitive cells, which correlated with apoptosis rates (Figure 4B). In B lympho- cytes and in tumor resistant cells, however, p17AE2 treat- ment did not affect pHi values. Nevertheless, there was not a correlation between basal pHi values in the different tumor B-cell lines and response to p17AE2 (Figure 4C). To evaluate whether the Cl−/HCO − exchange activity of AE2
removal of extracellular Cl−, and the pHi changes with and without p17AE2 were measured at different time inter- vals; under these forced conditions, a reversed exchange activity of AE2 is reflected by an increase in pHi (see Methods for details). In the sensitive cell line Jeko1, p17AE2 induced an increase in pHi with respect to cells incubated in the absence of the peptide, while in the resistant cell line U266 the activity of AE2 remained unchanged (Figure 4 D-E). These results suggest that in sensitive cell lines, like Jeko1, p17AE2 is able to modulate AE2 activity, alter- ing pHi and thus inducing cell apoptosis, while in resistant cell lines, such as U266, the peptide would not be able to alter AE2 activity and pHi would remain stable, not com- promising cell viability. Hence, in sensitive cell lines, p17AE2 would activate the physiological Cl−/HCO −
3
exchange function of AE2, favoring HCO − export in 3
exchange with Cl−, thus reducing pHi and promoting apoptosis.
Peptides specifically targeting AE2 induce apoptosis in tumor
Therapeutic effect of AE2 targeting peptides in mouse xenograft models in vivo
Finally, we assessed the effects of targeting AE2 in vivo.
For that purpose, 5x106 cells from three sensitive cell lines
(Mantle cell lymphoma- UPN1, DLBCL-OCI-Ly7 and mul-
i values ranging from 7.4 to 7.8 (Figure 4A). The alkaline
line into Rag2 IL-2gc mice, due to its sensitivity in the in vitro model. When tumors reached a size of 100 mm3, 200 mg or 400 mg per day of p17AE2-HT or truncated peptides were injected intratumorally during 14 days. Mice treated with 400 mg of p17AE-HT exhibited reduction in tumor volumes with respect to those treated with 200 μg or with truncated peptide (Figures 5F-G). These results indicate that p17AE2-HT treatment moderately reduced tumor growth in vivo.
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was modulated by p17AE2, cells were subjected to
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tiple myeloma-L363) were injected subcutaneously in
Rag2-/-IL-2gc-/- immunodeficient mice. When tumors
achieved volumes of 100 mm3, mice received daily intra-
tumoral injection of p17AE2 or a truncated peptide (50 mg
each). However, no changes in tumor size between con-
trol and treated mice were observed after 14 days of treat-
ment, suggesting that p17AE2 effects could be limited by
a poor bio-availability and susceptibility to degradation by
proteases (data not shown). Peptide cyclization has been
used as a strategy for stabilizing small peptides.49
Therefore, based on computational studies, we used this
strategy to design and synthesize two macrocyclic pep-
tides, one with a head-to-tail cyclization (termed p17AE2-
HT) and another one with a secondary amide as linker
(termed p17AE2-Amide) (Figure 5A). Surface plasmon res-
onance showed a dose-dependent binding of both
p17AE2-HT and p17AE2-Amide peptides to the third
extracellular loop of human AE2 (Figure 5B). However,
only the p17AE2-HT peptide, but not the p17AE2-Amide,
showed longer half-life and reduced clearance with
respect to the linear p17AE2 peptide (Figure 5C). In addi-
tion, p17AE2-HT decreased cell proliferation and promot-
ed apoptosis in different B-cell lymphoma cell lines and in
primary B-cell lymphoma samples, even more potently
than the p17AE2 linear peptide (Figures 5D-E). These
results indicated that p17AE2-HT was more efficient than
linear peptides and hence a good candidate for therapeutic
testing in vivo. To assess the therapeutic role of the macro-
cyclic peptides in vivo, we inoculated subcutaneously
In summary, our results suggest that targeting AE2 might exert a dual therapeutic effect in B-cell malignan- cies. In our in vitro studies, the linear p17AE2 was able to induce apoptosis of tumor B cells, while potentially boost- ing anti-tumor immune responses by reducing the number of Treg cells. In in vivo models, however, the linear peptide had no effect, probably because of its low stability, while the head-to-tail cycled p17AE2-HT showed increased sta- bility and managed to reduce tumor growth in vivo when given at a high dosage. Thus, AE2 seems to be a promising target in different B-cell malignancies, and modifications of AE2 targeting peptides may increase their potential therapeutic value in vivo.
Acknowledgments
We thank Elena Ciordia and Eneko Elizalde (CIMA) for excel- lent animal care.
Funding
The work was supported by grants from Fundación Ramón Areces, BAYER Pharma HealthCare (Grant4Targets 2015),
5x106 cells from the UPN1 B-cell lymphoma-derived cell -/- -/-
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