IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Disrupting inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP3R-derived peptide (TAT-IDPS)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca2+ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP3R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDPS in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDPS with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDPS to promote IP3R-mediated Ca2+ release. Although total IP3R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP3R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDPS-induced Ca2+ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP3R2-protein level, but not with IP3R1-, IP3R3- or total IP3R-expression levels. Inhibiting or knocking down IP3R2 activity in SU-DHL-4-reduced TAT-IDPS-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP3R2 and to promote IP3-induced pro-apoptotic Ca2+ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP3R hyperactivity. In particular, cancer cells expressing high levels of IP3R2 are addicted to IP3R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca2+ signaling and cell death.

IP 3 R-mediated Ca 2 þ signals with pro-apoptotic properties. 20 As a result, TAT-IDP-treated T lymphocytes displayed an increased sensitivity toward pro-apoptotic stimuli (like strong T-cell-receptor stimulation). Furthermore, we also developed a stabilized, protease-resistant form of the peptide (TAT-IDP DD/AA , which will be further indicated as TAT-IDP S ). TAT-IDP S provoked pro-apoptotic Ca 2 þ signals in CLL patient cells. 21 Hence, in contrast to normal cells, which were resistant to TAT-IDP S by itself but displayed enhanced sensitivity toward apoptotic triggers, CLL patient cells underwent apoptotic cell death in the presence of the peptide alone. This raises the question whether cancer cells, in particular Bcl-2-dependent malignancies, displayed altered Ca 2 þ -signaling properties that turned these cells into vulnerable targets toward peptides disrupting Bcl-2-mediated suppression of apoptotic IP 3 R activity. Importantly, these peptides selectively target the BH4 domain of Bcl-2, but not that of Bcl-Xl. 22 Here, we studied a set of cell lines derived from DL-BCL tumors, a disease characterized by its heterogeneity in gene expression, oncogenic aberrations, intrinsic apoptotic escape routes, and response to chemotherapy. [23][24][25] In particular, we focused on BH3-profiled 'primed to death' DL-BCL cell lines that are dependent on Bcl-2 upregulation. 26 We found that the relative IP 3 R2-expression level was an important determinant for the apoptotic response of these cells, and correlated with the ability of TAT-IDP S to trigger pro-apoptotic IP 3 R-mediated Ca 2 þ release. We found that disrupting Bcl-2 binding to IP 3 Rs was particularly effective in cancer cells with high levels of IP 3 R2. The presence of IP 3 R2 rendered these cells vulnerable toward ongoing IP 3 signaling, for example, like during chronic activation, whereas cells expressing relatively low levels of IP 3 R2 were much less sensitive. Such a correlation was not observed for the two other IP 3 R isoforms (IP 3 R1 and IP 3 R3). Hence, this is the first study to reveal a prominent role for the type of IP 3 R isoform as a determinant for the sensitivity to cell death in Bcl-2-dependent cancer cell lines.

Results
Some types of 'primed to death' DL-BCL cells are sensitive to TAT-IDP S . In a primary screening, we investigated five well-characterized and 'primed to death' DL-BCL lines, previously BH3 profiled by the laboratory of Dr. A. Letai (KARPAS422, TOLEDO, PFEIFFER, SU-DHL-4, and OCI-LY-1). 6,12 KARPAS422, TOLEDO, SU-DHL-4, and OCI-LY-1 display upregulated Bcl-2 levels and high amounts of Bcl-2/ Bim complexes, rendering these cells particularly sensitive toward the BH3-mimetic drug ABT-737, 12 whereas PFEIF-FER displayed relatively high levels of Bfl-1 and myeloid-cell leukemia 1 (Mcl-1) levels making this cell line more resistant to ABT-737. 12 Strikingly, we found remarkable differences in the response of these DL-BCL cells toward TAT-IDP S exposure (10 mM, 24 h) in cell-death experiments based on annexin V-FITC/propidium iodide (PI) staining and FACS analysis (Figures 1a and b). Indeed, in contrast to its scrambled counterpart, TAT-Ctrl (Supplementary Figure 1), TAT-IDP S triggered apoptotic cell death in four out of five DL-BCL cells (KARPAS422, TOLEDO, PFEIFFER, and SU-DHL-4), but not in OCI-LY-1. The latter was not due to a general defect in the apoptotic program in OCI-LY-1, because these cells were very sensitive toward more general apoptotic inducers, like staurosporine ( Figure 1c).
TAT-IDP S effectively provokes cell death in SU-DHL-4 but not in OCI-LY-1. Next, we decided to elucidate the underlying mechanisms for the different responses toward TAT-IDP S treatment. We focused on comparing SU-DHL-4 and OCI-LY-1, because these cells are both germinal-center DL-BCL cells and are very similar in cell size (Figure 2a). Furthermore, both cell lines expressed similar total amounts of IP 3 R proteins (Figure 2b), whereas displaying the most divergent response to TAT-IDP S . We first determined a concentration-response curve for both cells toward TAT-IDP S -induced cell death (Figure 2c). We found that TAT-IDP S killed SU-DHL-4 cells with an IC 50 of about 10 mM, whereas OCI-LY-1 cells were resistant to TAT-IDP S -induced cell death, even at 30 mM, which killed about 90% of the SU-DHL-4 cells. Using FITC-labeled TAT-IDP S , we also confirmed that both cell lines accumulated the peptide to similar extents (Supplementary Figure 2).
TAT-IDP S triggers IP 3 R-mediated cytosolic [Ca 2 þ ] rises in SU-DHL-4 but not in OCI-LY-1. Next, we monitored cytosolic Ca 2 þ signals in response to acute TAT-IDP S exposure in a Ca 2 þ -free extracellular medium ( Figure 3). After Fura2-AM loading of SU-DHL-4 and OCI-LY-1, cytosolic [Ca 2 þ ] measurements were performed in cell populations using an automated fluorescence plate reader. We found that TAT-IDP S (10 mM), but not TAT-Ctrl, caused an accelerated increase in the cytosolic [Ca 2 þ ] in SU-DHL-4, whereas this was not observed in OCI-LY-1 (Figure 3a). The increase in cytosolic [Ca 2 þ ] could be counteracted by using the selective IP 3 R antagonist xestospongin B (XeB), 27 indicating a major role for IP 3 Rs in mediating the TAT-IDPinduced [Ca 2 þ ] rise in SU-DHL-4 ( Figure 3b). Indeed, XeB reduced the slope of the TAT-IDP S -induced [Ca 2 þ ] rise by about 40%. These observations were underpinned by additional experiments in which the ER Ca 2 þ content was assessed using 10 mM thapsigargin, a potent and selective inhibitor of the ER Ca 2 þ ATPases (SERCA) 28 together with EGTA for chelating extracellular Ca 2 þ . The magnitude of the thapsigargin-induced [Ca 2 þ ] rise (area under the peak) is a measure of the amount of Ca 2 þ stored in the ER. We found that pretreating the cells with TAT-IDP S , but not TAT-Ctrl, severely reduced the thapsigargin-induced [Ca 2 þ ] rise in SU-DHL-4. In contrast, TAT-IDP S pretreatment had only a slight effect on the thapsigargin-induced [Ca 2 þ ] rise in OCI-LY-1 as compared with SU-DHL-4 ( Figure 3c). Importantly, all these measurements were done well before apoptotic cell death was observed, indicating that the acute rise of [Ca 2 þ ] was not due to ongoing apoptotic processes, but was rather a very proximal event in the induction of apoptosis. This is supported by the fact that treating SU-DHL-4 with BAPTA-AM (10 mM), a cell-permeable Ca 2 þ chelator, reduced TAT-IDP S -induced cell death by B75% (Figure 4a). Similar results were observed for XeB (2.5 mM), which inhibits IP 3 R-mediated Ca 2 þ release. Indeed, a 2-h pre-treatment of SU-DHL-4 with XeB reduced the number of apoptotic cells (i.e., annexin V-FITC-positive cells) in response to TAT-IDP S by about 40% in comparison to SU-DHL-4 treated with TAT-  Figure 3b). XeB at 2.5 mM did not completely prevent apoptosis, probably because IP 3 R signaling was not completely blocked in these experimental conditions. Higher concentrations of XeB could, however, not be used because they seemed toxic to these cells (data not shown). This effect may be related to the important role of IP 3 Rs in autophagy, which is important for cancer cell survival. 29,30 Both SU-DHL-4 and OCI-LY-1 display upregulated Bcl-2, but express different IP 3 R isoforms. We quantified the expression levels of a variety of anti-apoptotic Bcl-2-family members and IP 3 R isoforms in both SU-DHL-4 and OCI-LY-1 at the mRNA level using specific probes ( Figure 5, bar graphs), and at the protein level using specific and validated antibodies ( Figure 5, blots). As reference cell lines, we used HT cells, a DL-BCL cell line, which has very low endogenous levels of Bcl-2, and HT cells ectopically and stably overexpressing Bcl-2 (HT-Bcl-2). We found that both SU-DHL-4 and OCI-LY-1 displayed similar levels of Bcl-Xl ( Figure 5a) and Mcl-1 ( Figure 5b). These levels were also similar to the levels found in HT and HT-Bcl-2, although Bcl-Xl was slightly higher in both SU-DHL-4 and OCI-LY-1. For Bcl-2, we also found a high expression in both SU-DHL-4 and OCI-LY-1. Its level was in the range of the Bcl-2-overexpressing HT ( Figure 5c). However, Bcl-2 levels were significantly higher in OCI-LY-1 than in SU-DHL-4.
We also probed IP 3 R isoform-expression levels by qRT-PCR and by western blot analysis using isoform-specific antibodies. We also found that both SU-DHL-4 and OCI-LY-1 displayed similar levels of IP 3 R1, which were slightly higher than the ones observed in HT and HT-Bcl-2 ( Figure 5d). However, SU-DHL-4 and OCI-LY-1 displayed a very different profile for IP 3 R2 (Figure 5e) and IP 3 R3 (Figure 5f). Indeed, SU-DHL-4 displayed a strong upregulation of IP 3 R2-mRNA and -protein levels as compared with the other cell lines, whereas OCI-LY-1 displayed the highest IP 3 R3-mRNA andprotein levels.
Given the striking difference in IP 3 R-expression profile and the distinct sensitivity of these isoforms toward IP 3 , we wondered whether high IP 3 R2 expression was underlying the sensitivity of DL-BCL cells toward TAT-IDP S exposure. Importantly, IP 3 R2 and IP 3 R3 have very distinct ligand sensitivity: IP 3 R2 is the IP 3 R isoform most sensitive toward IP 3 , whereas IP 3 R3 is the IP 3 R isoform least sensitive toward IP 3 . 31 Therefore, we developed a siRNA probe selectively targeting IP 3 R2 and a non-targeting control siRNA probe (siCtrl). This siRNA probe (siIP 3 R2) effectively knocked down IP 3 R2-protein levels in transfected SU-DHL-4 cells by about 60% in comparison to mock-transfected or siCtrl-transfected SU-DHL-4 cells (Figure 6a, blots). IP 3 R2 knockdown correlated with an increased resistance toward TAT-IDP S treatment (Figure 6a, bottom panels). Indeed, the siIP 3 R2 probe significantly reduced the number of apoptotic cells in TAT-IDP S -treated SU-DHL-4 in comparison to mock-transfected or siCtrl-transfected SU-DHL-4 ( Figure 6a, bottom panel). Conversely, when we increased the expression of IP 3 R2 in OCI-LY-1 cells by the transfection of an IP 3 R2-expression plasmid, OCI-LY-1 became more sensitive toward apoptotic   (Figure 7a, right panel). We also performed an IP 3 R-profile analysis for the four DL-BCL cell lines using a pan-IP 3 R antibody recognizing all three IP 3 R isoforms and using isoform-specific antibodies (Figure 7b, blots, left panels). Plotting the apoptotic responses (Figure 7b, central panels) and [Ca 2 þ ] responses (Figure 7b, right panels) to TAT-IDP S as a function of the different IP 3 R isoforms and total IP 3 R-protein levels for the different DL-BCL cell lines revealed that only IP 3 R2-protein levels, but not IP 3 R1, IP 3 R3, nor total IP 3 R, correlated with TAT-IDP S -induced apoptosis (r 2 ¼ 0.7) or with  Figures 8a and b).
We have previously shown that the Bcl-2-binding site is conserved among all three IP 3 R isoforms. 22 At least in in vitro surface-plasmon-resonance experiments, recombinantly expressed and purified fragments covering the proposed Bcl-2-binding site of IP 3 R1, IP 3 R2, and IP 3 R3 were able to interact with the synthetic BH4 domain of Bcl-2. 22 Thus, we examined whether this was also valid in a cellular context, and whether Bcl-2 co-immunoprecipitated with IP 3 Rs from SU-DHL-4 and OCI-LY-1 cell lysates. Immunoprecipitation of IP 3 R2 indeed caused the co-immunoprecipitation of Bcl-2 in both SU-DHL-4 and OCI-LY-1 lysates. However, despite the fact that OCI-LY-1 displayed higher levels of Bcl-2 than SU-DHL-4, the amount of Bcl-2 that was specifically co-immunoprecipitated with IP 3 R2 in OCI-LY-1 was extremely low. Importantly, we found that pretreatment of SU-DHL-4 with TAT-IDP S reduced the amount of Bcl-2 coimmunoprecipitating with IP 3 R2 (Figure 8a). A similar band was observed in OCI-LY-1, but due to the much lower levels of Bcl-2 binding to IP 3 R2 it was just above the detection level and this was despite the very high Bcl-2 levels in these cells.
For IP 3 R3, we found that only in OCI-LY-1, but not in SU-DHL-4, Bcl-2 co-immunoprecipitated with IP 3 R3. Pretreatment with TAT-IDP S only slightly reduced Bcl-2 levels in the IP 3 R3 co-immunoprecipitated samples (Figure 8b). Hence, these experiments indicate that in SU-DHL-4 Bcl-2 was recruited to a large extent by IP 3 R2, and Bcl-2 could be This was not observed in OCI-LY-1 with respect to the predominant IP 3 R3 isoform in these cells. This could mean that the Bcl-2/IP 3 R3 interaction is less pronounced in a cellular context or alternatively that Bcl-2 in these cells is mainly bound to other proteins such as Bim and Bax. 12 Thus, these observations suggest that the TAT-IDP S -induced [Ca 2 þ ] rise and cell death are linked to the disruption of the IP 3 R/Bcl-2 interaction, particularly in cells expressing relatively high levels of IP 3 R2.

Discussion
The major findings of this study are that (i) IP 3 R2 is a determinant of the sensitivity of Bcl-2-dependent 'primed to death' DL-BCL cells toward the apoptotic effect of TAT-IDP S , and (ii) Bcl-2-dependent cancer cells may be addicted to high levels of Bcl-2 to suppress aberrant pro-apoptotic Ca 2 þ signals. In particular, cancer cells expressing the most sensitive IP 3 R isoform (IP 3 R2) likely are very vulnerable toward tonic IP 3 signaling.   Peptide tools selectively targeting BH4-Bcl-2 are effective in DL-BCL cancer cells expressing high levels of IP 3 R2. Our study is the first to provide a prominent role for distinct IP 3 R isoforms in cell death and survival processes in malignant cells. The higher IP 3 sensitivity of IP 3 R2 could render cells sensitive to very low levels of IP 3 . In that respect, TAT-IDP S may trigger Ca 2 þ -release events by disrupting Bcl-2/IP 3 R2 interactions, in conditions of low-level stimulation and close to basal cellular IP 3 concentrations. These events may not be sufficient to trigger activation of the least sensitive IP 3 R isoform, the IP 3 R3. This would render cancer cells expressing mainly IP 3 R3 resistant to TAT-IDP S . At the molecular level, the sensitivity toward TAT-IDP S is reflected in the presence of different Bcl-2/protein complexes. Indeed, the very sensitive SU-DHL-4 displayed high levels of IP 3 R/ Bcl-2-complex formation, whereas this was not the case for the resistant OCI-LY-1, although this cell line expressed even higher levels of Bcl-2 than SU-DHL-4. Our observation is fully in line with a previous report showing that OCI-LY-1 displayed high levels of Bcl-2/Bax complex formation, which was not the case for SU-DHL-4. 12 Hence, it seems that dependent on the apoptotic escape route cells may be addicted to high levels of Bcl-2 either to suppress aberrant IP 3 R activity (like in the case of IP 3 R2-expressing cancer cells, e.g., SU-DHL-4) or to suppress aberrant Bax activity (like in the case of IP 3 R3-expressing cancer cells, e.g., OCI-LY-1). Indeed, although both IP 3 R isoforms may interact with Bcl-2 in vitro, the occurrence and significance of these interactions may be very different in a cellular context. Therefore, it may be less critical for cancer cells to use Bcl-2 for suppressing the activity of IP 3 R3, because this isoform is the least sensitive to IP 3 and thus to ongoing B-cell receptor (BCR) signaling. In contrast, cancer cells expressing high levels of IP 3 R2 will be addicted to high levels of Bcl-2 to suppress the pro-apoptotic activity of the hypersensitive IP 3 R2 in response to ongoing IP 3 signaling. Interestingly, it has been shown that DL-BCL cells have a chronically active BCR. 32 Moreover, SU-DHL-4 and OCI-LY-1 are reported to have a similar moderate activation of PLCg2. 33 This may indicate that cancer cells may suppress the downstream effects of chronic BCR signaling by either Bcl-2/IP 3 R interactions to inhibit IP 3 R signaling or alternatively by switching to the less sensitive IP 3 R3 isoform. From our immunoprecipitation experiments, it was evident that TAT-IDP S did not completely disrupt the binding of Bcl-2 to IP 3 Rs. This may be due to the fact that other Bcl-2 domains may contribute to IP 3 R binding. 34 Yet, alternative mechanisms could be involved in the differential role of different IP 3 R isoforms in cell death. It has recently been shown that the phosphorylation of IP 3 R3 by Akt leads to diminished Ca 2 þ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt. 35 Therefore, we cannot exclude an implication of Akt-induced phosphorylation of IP 3 R3 in the resistance of cells that highly express IP 3 R3 (OCI-LY-1) toward TAT-IDP S induction of apoptotic Ca 2 þ signals, rendering Bcl-2 proteins redundant for recruitment to the IP 3 R3 channels.
Novel functions for IP 3 R2 in cancer cells beyond its canonical function in exocrine glands. This study also reveals a novel isoform-specific function for IP 3 R2. Although IP 3 R2 is expressed at very low levels in most tissues, it is highly expressed in organs with exocrine functions, correlating with its importance for the physiological exocrine function of these organs. 36 IP 3 R2 cooperates with IP 3 R3 in nutrient digestion and enzymatic secretion, correlating with severely impaired Ca 2 þ signaling in double knock outs in acinar cells of the salivary glands and of the pancreas 37 and in olfactory mucus secretion and function. 38 In acinar cells, IP 3 R2 expression levels have been linked to the sensitivity toward metabolic stress, as IP 3 R2 is the most sensitive toward ATP regulation and determines the influence of ATP depletion on intracellular Ca 2 þ signaling. 39 Here, we describe for the first time a prominent role for IP 3 R2 for the pathophysiology of B-cell lymphoma malignant cells. The aberrant IP 3 R2 upregulation in some B-cell cancer cells may be an additional component in their addiction to high levels of Bcl-2 to suppress toxic Ca 2 þ signals in response to chronic BCR signaling, adding another level of heterogeneity of these cancer cells toward dysregulation of apoptosis-signaling cascades. The mechanism underlying IP 3 R2 upregulation is not clear, but clearly is a transcriptionally regulated event. Also, the benefit for cancer cells to upregulate IP 3 R2 is not clear. Nevertheless, given the central role of constitutive IP 3 / Ca 2 þ signaling in regulating mitochondrial bio-energetics, 40 IP 3 R2 upregulation may enhance mitochondrial function and energy production to accommodate for the higher metabolic activity and the induced proliferation of cancer cells.

Conclusion
Our findings highlight the importance of targeting Bcl-2's BH4 domain in Bcl-2-dependent cancers. Although we previously showed that CLL may be targeted using IP 3 R-derived peptides, we now provide (i) evidence that this strategy is applicable in other cancer cells like DL-BCL, and (ii) mechanistic insights in the underlying signaling pathways revealing a prominent role for IP 3 R2. This strategy may be helpful to sensitize cancer cells to BH3-mimetic drugs, including cancer cells that are resistant to TAT-IDP S itself. It also seems that exploiting the adaptive response of cancer cells toward higher metabolic needs putatively underlying IP 3 R2 upregulation may provide a novel way to target these cells through Ca 2 þ -signaling dysregulation.