Tetraspanin CD37 Directly Mediates Transduction of Survival and Apoptotic Signals

Abstract 622

CD37 is a tetraspanin selectively expressed on normal and transformed B-cells with unknown function. A role for CD37 in signal transduction pathways affecting cell development and activation has been proposed. However, the direct involvement of CD37 in signaling has always been excluded due to short cytoplasmic tails that lack canonic signaling motifs. Given its B-cell selectivity CD37 is a candidate therapeutic target for B-cells malignancies including chronic lymphocytic leukemia (CLL). Small Modular ImmunoPharmaceutical proteins targeting CD37 such as SMIP-016 have already demonstrated clinical activity and yet their mechanism of killing has not been described. Despite the common belief that tetraspanins only act as “molecular facilitators”, with no direct role in signal transduction, we demonstrate a completely new role for CD37. Analysis of CD37 sequence revealed the presence of two tyrosine residues in cytoplasmic tails, Tyr¹³ within a predicted weak ITIM-motif, (known to recruit inhibitory signaling effectors such as SHP1) and Tyr²⁷⁴ within a single tyrosine ITAM-like motif. To study the relevance of each of these tyrosine residues to SMIP-016 induced cell death we generated 697 cell lines expressing wild type or mutant Flag-tagged CD37 [Tyr¹³(−) or Tyr²⁷⁴(−)] and demonstrated by biochemical and proteomic analysis that upon ligation CD37 becomes phosphorylated, associates to the membrane lipid rafts, recruits pLyn and pSHP1 at the Tyr¹³ and initiates a cascade of events leading to mitochondrial membrane depolarization (MMD) and apoptosis. Down regulation of SHP1 by siRNA or Sodium Stibogluconate (10ug/ml) resulted in almost complete loss of SMIP-016-induced cytotoxicity (p<0.0001) strongly supporting the involvement of pSHP1 in CD37 mediated signaling. Furthermore we demonstrated that Tyr¹³ is essential for the transduction of death signals since the lack of it but not Tyr²⁷⁴ prevented SMIP-016 induced phosphorylation of CD37, association with SHP1 and cell death. In the setting of late B-cell activation, BIM is a critical pro-apoptotic protein responsible for mitochondrial membrane stabilization and apoptosis. Interestingly, CD37 ligation of CLL cells, resulted in consistent FoxO3a dependent transcription of Bim mRNA (p<0.001) as demonstrated by ChIP, luciferase and pull down assays while level of other pro- and anti-apoptotic proteins such as BAX, BCL2 and MCL1 remain unchanged. BIM down-modulation by siRNA resulted in inhibition of depolarized mitochondria (p=0.0025) with increase in intact mitochondrial integrity (p=0.002) and in the reduction of SMIP-016-induced cytotoxicity compared to scrambled control (p<0.001) confirming the involvement of BIM in SMIP-016-mediated MMD and apoptosis. Proximal SHP1 activation by SMIP-016 directly contributes to BIM induction, since the down modulation of SHP1 by siRNA antagonized BIM up-regulation in CLL cells. Moreover lack of Tyr¹³ but not Tyr²⁷⁴ prevented SMIP-016 induced up-regulation of BIM further indicating that Tyr¹³ is essential for the induction of cell death. Interestingly, the deletion of Tyr²⁷⁴ resulted in increased SMIP-016 induced cytotoxicity (p<0.0001) indicating a negative regulatory function of this region in CD37 mediated cell death, consistent with the presence of an ITAM motif in this domain. The p85 regulatory and the p110 catalytic subunits of phosphatidylinositol 3-kinase (PI3K) have been shown to bind to phosphorylated tyrosines within the ITAM motif in other cell types. By co-immunoprecipitation studies we demonstrated that Tyr²⁷⁴ negatively regulates cell death by activating a PI3K dependent survival loop by recruitment of p85 and p110δ and downstream phosphorylation of GSK3β. In addition, treatment of cell lines expressing wild type CD37 or CLL cells with the PI3K inhibitor LY294002 blocked PI3K phosphorylation and resulted in increased SMIP-016 mediated cell death. In conclusion, this dual direct signaling role of a tetraspanin protein has not been previously described. The findings put forth provide rationale for therapeutic antibodies or peptides which selectively activate Tyr¹³. They also provide strong rationale for combination of SMIP-016 with PI3-kinase inhibitors which have been shown by our group and others promising therapeutic option for CLL.