Regulation of CD47-mediated cell death by p21-activated kinases

Regulation of CD47-mediated cell death by p21-activated kinases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Regulation of CD47-mediated cell death by p21-activated kinases Chinten Lim, Pascal Leclair, Anuli Uzozie, Amanda Lorentzian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8940785/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract CD47, a cell-surface glycoprotein widely recognized as a "don't eat me" signal, also serves as a receptor capable of triggering caspase-independent cell death. While actin dynamics have been implicated in this process, the upstream signaling regulators remain poorly defined. In this study, we identify p21-activated kinases (PAKs) as critical negative regulators of CD47-mediated cell death in acute lymphoblastic leukemia (ALL). Using phospho-proteomic and Inferred Kinase Activity (INKA) analysis, we observed that CD47 ligation with the monoclonal antibody CC2C6 leads to the downregulation of actin-regulatory kinases, including PAK1, PAK2, and PAK4, in patient-derived xenograft (PDX) models and Jurkat T-ALL cells. Pharmacological inhibition of PAKs using the pan-PAK inhibitor PF-3758309 markedly synergized with CD47-antibody CC2C6 to induce cell death across multiple B- and T-ALL cell lines. This synergy extended to inhibitors of other actin regulators, including ROCK, PKD, and the Rho-family GTPases, Cdc42 and Rac1. Mechanistically, we demonstrate that hypersensitivity to CD47 ligation in a novel Jurkat substrain (Jkt75) correlates with reduced basal PAK activity and increased levels of active (dephosphorylated) cofilin. We confirm that CD47-mediated death requires dynamic F-actin remodeling, as both the actin depolymerizer cytochalasin D and the stabilizer jasplakinolide significantly attenuated cell death. Furthermore, we reveal that CD47 ligation triggers robust cell-cell aggregation, which is actin-dependent and essential for the lethal signal; preventing physical cell-cell contact through rotation or immobilization effectively abolished the death response. Our findings establish a novel PAK-actin-aggregation axis that governs CD47-mediated programmed cell death. These results suggest that targeting PAK signaling may provide a potent strategy to enhance the efficacy of CD47-based immunotherapies in refractory leukemias. Health sciences/Diseases/Cancer/Paediatric cancer Biological sciences/Cell biology/Cell adhesion CD47 cell death PAK F-actin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction CD47 is a plasma membrane penta-spanning cell-surface glycoprotein best known as a ‘don’t eat’ anti-phagocytic signal upon engaging SIRPα, its counter receptor expressed on professional phagocytes [ 1 ]. Via its association with integrins, CD47 is also involved in cell adhesion and migration. However, a lesser studied role for CD47 is its ability to induce cell death in a variety of cells [ 2 ]. Early investigations into CD47 revealed its role in inducing a rapid onset of cell death when ligated by antibodies or its natural ligand, thrombospondin (TSP). The features of CD47-mediated cell death include phosphatidylserine (PS) exposure and mitochondrial abnormalities, such as ROS production and decreased mitochondrial membrane potential. However, in contrast to classical apoptosis, CD47-mediated cell death (at one time coined as ‘type III’) is caspase-independent, does not involve cytochrome c release from mitochondria, nor does it involve nuclear alterations [ 3 , 4 , 5 ]. We reported previously the CD47 antibody clone CC2C6 efficiently induced cell death in solution, in contrast to clone B6H12 which requires immobilization [ 6 , 7 , 8 , 9 ]. Prior studies have identified a role for actin dynamics in modulating CD47-mediated cell death, as exemplified by the inhibitory effects of the actin depolymerizer, cytochalasin D [ 3 , 5 , 6 , 10 ]. Furthermore, Mateo et al. found that cells isolated from Wiskott-Aldrich syndrome (WAS) patients, who have abnormal expression of WAS protein (WASp, an actin nucleation-promoting factor of the Arp2/3 complex) were insensitive to CD47-mediated cell death [ 6 , 11 ]. Microscopy based studies also showed alterations in filamentous actin following CD47 ligation [ 12 ]. Actin involvement in CD47 processes is not limited to cell death but has also been shown to increase CD47 homophilic cell aggregation in fibroblasts [ 13 ], stabilize CD47 at the cell membrane of erythrocytes [ 14 ], and induce the formation of stress fibers [ 15 ] or cortical bundles [ 12 ]. In addition, CD47 colocalized with cortical actin, E-cadherin, and ZO-1 at cell-cell-contacts of epithelial cells [ 16 ], and with fascin, an actin bundling protein, at the tips of microspikes generated by COMP ( i.e. , TSP-5) binding to CD47 in ligament cells [ 17 ]. Finally, CD47 ligation has been shown to induce the depolymerization and degradation of actin [ 3 , 5 , 6 , 10 ], while others have shown an increase in F-actin following CD47 ligation, a phenomenon that was amplified by CD3 ligation [ 18 ]. Actin dynamics are regulated by G proteins via a variety of intermediaries, such as p21-activated kinases (PAKs), cofilin, Arp2/3, and WASp [ 19 ]. The PAK family of proteins are distinguished by their regulatory domain and mechanism of activation. Binding of activated small GTPases to group I PAKs (PAK1-3) induce a conformational change that enables their phosphorylation and activation. In contrast, group II PAKs (PAK4-6) are constitutively phosphorylated at the equivalent site and their activation seems to be determined solely on the binding of small GTPases or an SH3 domain, though the precise mechanism is still under investigation [ 20 , 21 , 22 ]. Nonetheless, activation of PAKs leads to phosphorylation and activation of LIMK, which in turn leads to phosphorylation and inactivation of cofilin [ 23 ]. Cofilin regulates actin dynamics by binding and severing F-actin, which initiates actin re-assembly into various structures depending on the different proteins present. For example, WASp and the Arp2/3 complex are involved in building branched networks in lamellipodia, whereas fascin aids in forming parallel bundles in filopodia [ 24 ]. Furthermore, the actin cytoskeleton is also important in establishing and maintaining various types of cell-cell junctions, including immunological synapses [ 25 ]. Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, consisting of precursor B and T subtypes (B-ALL and T-ALL) occurring at a 17:3 ratio. Although the 5-year survival rate for childhood leukemia now approaches 90%, up to 20% of patients relapse at least once and require treatment intensification [ 26 , 27 ]. Importantly, the highly toxic drugs administered to children undergoing chemotherapy at any stage result in serious long-term sequelae, including developmental defects, cardiotoxicities, neurotoxicities, and secondary malignancies [ 28 ]. As such, the development of novel adjuvant or second-line therapies that enable reduced dosing of conventional cytotoxic agents continue to be needed [ 29 ]. Here, we extend the findings of the actin cytoskeleton as a downstream regulator of CD47-mediated cell death in ALL and show that a synergistic increase in CC2C6-mediated cell death can be achieved by inhibition of PAKs upstream of cytoskeleton dynamics. Furthermore, we show that a derivative Jurkat cell line exhibits very high levels of CD47-mediated cell death that is correlated with reduced activated PAKs and increased F-actin compared to its isogenic parental strain. Finally, we show that actin-mediated cell aggregation is required for maximal sensitivity to CC2C6. Results PAK signaling regulates CD47-mediated cell death. Previously, we showed that treating Jurkat T-ALL cells with CC2C6 induces significant cell death in as little as 30-minutes, with maximal effect observed between 2–6 hours [ 7 ]. However, this response is not consistent across a number of ALL cell lines tested, including in patient-derived xenografts (PDX) of B- and T-ALLs. For example, in T-ALLs, BT009 showed no significant response and BT012 only a marginal one (Fig. 1 A). Of note, multiple PDXs of BT007, which included leukemic blasts from diagnosis (Dx), at end-of-consolidation (D29) and at relapse (R1), exhibited comparable and conserved response to CC2C6 treatment (Fig. 1 A). In B-ALLs, BB007, BB013, BB039 and BB050 showed minimal to no response to CC2C6 (Fig. 1 B). Of particular significance, the relapsed tumor BB026-R1 exhibited almost a two-fold increase in CC2C6-induced cell death when compared to the diagnostic tumor BB026-Dx (Fig. 1 B, C). Relapsed leukemias typically exhibit increased resistance to standard treatments like doxorubicin and vincristine, a phenomenon also observed for BB026-R1 when compared to BB026-Dx ( Supp Fig. 1 ). The levels of cell surface expressed CD47 was comparable between BB026-Dx and -R1 (Fig. 1 D), suggesting the difference in CC2C6-induced cell death is likely due to differences in cell intrinsic signaling unrelated to CD47 levels. Rationalizing that differences in kinase activities may underlie the differential sensitivity of BB026-Dx and -R1 cells to CD47-ligation, we performed phospho-proteome analysis of cells without and with CC2C6 treatment (Fig. 2 A, B). Using Inferred Kinase Activity (INKA) analysis [ 30 ], we noted a number of kinases with actin regulatory functions were downregulated following CD47-ligation, including ROCK1, ROCK2, PAK1, PAK2 and PAK4 ( Supp Fig. 2 ). To understand the role of PAKs in CD47-mediated cell death, we treated cells with CC2C6 and the pan-PAK inhibitor, PF-3758309 [ 31 ]. Co-treatment of CC2C6 and PF-3758309 for 2 hrs resulted in increased cell death occurring in a synergistic manner for BB026-Dx, but not for BB026-R1 (Fig. 3 A). Consistent with our prior findings [ 7 ], treatment with CC2C6 for 24 hrs resulted in lower levels of cell death compared to 2 hrs, but also facilitated longer incubation with a secondary agent. In this manner, co-treatment of CC2C6 and PF-3758309 for 24 hrs resulted in increased and synergistic cell death observed for both BB026-Dx and -R1 cells (Fig. 3 B), indicating that PAK activity regulates CD47-ligation mediated cell death. We then assessed if this phenomenon could be more widely replicated in various leukemic cell lines. Synergistic activities were observed for Jurkat, MOLT-4 and BV-173 cells treated with CC2C6 and PF-3758309 for either 2 or 24 hrs (Fig. 3 C-E). Our testing was also extended to NALM6 cells, a B-ALL cell line that exhibited minimal CD47-ligation induced cell death in our standard 2 hr assay. At best, combined CC2C6 and PF-3758309 treatment for 2 hrs revealed only a small and additive effect, while a 24 hrs treatment revealed a clear synergistic effect on cell death (Fig. 3 F). Whenever possible, we selected a monoagent concentration of PF-3758309 that yielded a minimal effect on cell death for each assay condition (Fig. 3 C-F). It is notable that the observed synergistic effects remained evident for cells treated with CC2C6 and a higher concentration of PF-3758309 ( Supp Fig. 3 ). PF-3758309 is a potent inhibitor of group I and group II PAKs, including PAK1 and PAK4. To assess if CD47-mediated cell death may involve differential regulation by group I or II PAKs, we also tested the effect of PAK1 or PAK4 inhibition using the selective inhibitors, NVS-PAK1-1 and KPT9274 [ 32 , 33 ]. In Jurkat cells, CC2C6 had much lower synergistic effects with PAK1- or PAK4- selective inhibitors when compared with the pan-PAK inhibitor (Fig. 3 G-H), suggesting that this is not an isoform-specific phenomenon. Next, we assessed the role of proteins known to regulate PAK activity, including the Rho-family of small GTPases and protein kinase D (PKD)[ 22 ], the latter of which was also implicated in our INKA analysis ( Supp Fig. 2 ). The Cdc42 inhibitor, ML-141 [ 34 ], exhibited modest synergy with CC2C6 to induce Jurkat cell death after 2 and 24 hr treatments (Fig. 4 A). In contrast, synergistic activity was only observed following 24 hr co-treatment with the Rac1 inhibitor, EHop-016 [ 35 ] (Fig. 4 B). As for the pan PKD inhibitor, CID755673 [ 36 ], synergistic activity was only observed with 2 hr co-treatment (Fig. 4 C). Finally, we assessed the inhibition of the actin regulator, ROCK, since it was implicated in our INKA analysis and its function parallels that of PAK. As such we found synergy between the ROCK inhibitor, Y-27632 [ 37 ], and CC2C6 at both 2 and 24 hr incubations (Fig. 4 D). Taken together, our results reveal that CD47 ligation-induced cell death is enhanced by inhibition of PAK signaling and regulators of actin cytoskeletal remodeling. Expression and activity of PAK and cofilin in CD47-mediated cell death. Serendipitously, we derived a substrain of Jurkat cells, termed Jkt75, which consistently exhibited much greater levels of CC2C6-induced death, up to 75%, when compared to the ~ 25% observed in the parental cells (herein termed Jkt25, Fig. 5 A). Similar to our results using BB026 cells, INKA analysis of phospho-proteomic data of Jkt25 and Jkt75 cells following CC2C6 treatment revealed similar downregulated activity of PAKs ( Supp Fig. 4 ). To understand the role of PAKs in CD47-mediated cell death, we compared the expression of phosphorylated and total PAK1 and PAK4 with and without CC2C6 treatment (Fig. 5 B). Compared to Jkt25, Jkt75 cells expressed higher levels of PAK1, and lower levels of pT423-PAK1 and pS474-PAK4 (Fig. 5 C, D). CD47 ligation increased the expression of PAK1 in Jkt25 cells, but did not result in measurable change of pPAK1 or pPAK4 in either cell types. As a proxy for PAK activation, we also assessed if the downstream actin regulator, cofilin, might be differentially regulated after CC2C6 treatment. While total cofilin levels were comparable in both Jkt25 and Jkt75 cells, the inactive pS3-cofilin was significantly higher in Jkt25 (Fig. 5 E), consistent with the increased pT423-PAK1 and pS474-PAK4 observed. As the immunoblot-based assays provided limited resolution of changes induced by CC2C6 treatment, we utilized an intracellular flow cytometry-based assay for pS3-cofilin to enhance the temporal resolution of changes associated with actin cytoskeletal dynamics ( Supp Fig. 5 ). CC2C6 treatment of Jkt25 cells revealed a bi-phasic response in pS3-cofilin, with an initial decrease observed at 15mins and a second phase after 1 hr (Fig. 5 F). Taken together, these results suggest that increased sensitivity to CD47-mediated cell death is correlated with reduced PAK signaling and dynamic changes in cofilin activity. Actin cytoskeletal dynamics modulate CD47-mediated cell death. Previous studies have shown that CD47 ligation induces F-actin depolymerization, and that cytochalasin D (CytoD) treatment attenuates CD47-mediated cell death [ 3 , 5 , 6 , 10 , 38 ]. Given that PAK activity regulates the actin cytoskeleton and we observe increased cofilin activity following CC2C6 treatment, we assessed if CD47-ligation with CC2C6 also modulates the actin cytoskeleton. CD47-ligation with CC2C6 resulted in a significant decrease in F-actin in both Jkt25 and Jkt75 cells, with the latter exhibiting a more substantive decrease correlating with higher basal F-actin levels (Fig. 6 A). We observed that CC2C6 treatment resulted in even lower F-actin levels in Jkt75 cells compared to the most potent concentration of CytoD ( Supp Fig. 6 A), suggesting the involvement of distinct F-actin depolymerization mechanisms in Jkt75 cells. A significant decrease in F-actin levels following CC2C6 treatment was seen for B0026-R1 cells, but not for B0026-Dx cells (Fig. 6 B). We found that CytoD significantly attenuated cell death induced by CC2C6 in Jkt25, Jkt75, B0026-Dx, B0026-R1, MOLT4, and BV-173 cells (Fig. 6 C-F). Cell surface CD47 expression was unchanged with CytoD treatment ( Supp Fig. 6 B), hence the cell death inhibition observed was not attributed to decreased receptor function. Since PAK is a known up-stream regulator of actin dynamics, we tested if CytoD could negate the synergistic effects of combined CD47-ligation and PAK-inhibition. Indeed, cell death decreased by more than half in both Jkt25 and Jkt75 cells when CytoD was incubated along with CC2C6 and PF-3758309 (Fig. 6 G,H). We also found that CC2C6-induced cell death was attenuated in cells co-treated with jasplakinolide (Fig. 6 I), a binder of actin and F-actin stabilizer [ 39 ]. Given that both CytoD and jasplakinolide share the ability to disrupt actin function, we surmised that CD47-mediated cell death involves dynamic remodeling of the F-actin cytoskeleton. CD47-mediated cell death requires actin-mediated cell-cell contacts. We showed previously that CC2C6 treatment led to Jurkat cell-cell aggregation [ 7 ]. Given that F-actin is involved in reinforcing cell-cell contacts [ 40 ], we hypothesized that CD47-mediated cell death involves actin cytoskeletal remodeling and cell-cell aggregation. Indeed, Jkt75 cells exhibited greater CC2C6 treatment-induced aggregation when compared to Jkt25, with CD47 −/− cells showing no response (Fig. 7 A-C, Supp Fig. 7 ). Consistent with the requirement for F-actin cytoskeletal remodeling, co-incubation with CytoD and CC2C6 reduced the formation of aggregates for both Jkt25 and Jkt75 cells (Fig. 7 A,B). We also assessed if physical limitation of cell-cell contacts is sufficient to inhibit CD47-ligation induced cell death by incubating cells under constant vessel movement (rotation) or pre-immobilized on poly-L-lysine coated dishes. Under these conditions, we found that cell death was significantly inhibited for Jkt25 and Jkt75 cells treated with CC2C6 (Fig. 8 A,B). In sum, our cumulative data are consistent with the interpretation that CD47-ligation mediated cell death requires modulation of F-actin dynamics and cell-cell aggregation. Discussion PAKs as novel regulators of CD47-mediated cell death. We demonstrate for the first time that members of the p21-activated kinase (PAK) family regulate CD47-mediated cell death via the actin cytoskeleton. Initial phospho-proteomic analysis of PDX B-ALL cells suggested the activity of several PAKs were downregulated following treatment with CD47 antibody (clone CC2C6), which correlated with induction of cell death. This phenomenon was also observed in the Jurkat T-ALL cell line. Interestingly, while Western blot analysis revealed the hypersensitive Jkt75 substrain exhibited significantly lower baseline levels of active PAKs compared to Jkt25 cells, CC2C6 treatment did not further alter their PAK phosphorylation states. This discrepancy between proteomics and Western blot analysis may stem from the higher sensitivity of proteomic approaches compared to immunoblotting. Alternatively, the INKA algorithm infers kinase activity based on the phosphorylation state of downstream proteins [ 30 ]. It is therefore plausible that proteins involved in cytoskeletal dynamics other than PAKs were responsible for the change in protein phosphorylation assigned to PAKs by the algorithm. Indeed, INKA analysis indicated that the PAK regulator protein kinase D (PKD)—which also regulates actin dynamics via modulation of slingshot and cofilin independently of PAKs [ 19 ] —and ROCK, a kinase whose role in actin regulation is well established [ 41 ], were also downregulated in CC2C6-treated samples. Supporting this, inhibition of these kinases (PAKs, PKD and ROCK), or Rho GTPases (which act as PAK activators but also regulate actin independently), synergized with CC2C6 in our experiments to induce increased cell death. Actin dynamics and reorganization. The actin cytoskeleton is a dynamic regulator of cell shape and is the driving force of cell motility. Consistent with previous findings [ 3 , 5 , 6 , 10 , 38 ], we confirm that cell death induced by CC2C6 is partially inhibited, and in some cases, completely inhibited, by actin depolymerization using cytochalasin D. Notably, CytoD also abolished the synergy between CC2C6 and PAK inhibition, suggesting that an intact actin structure is required for CD47-mediated death. However, we also observed that the actin stabilizer jasplakinolide inhibited CD47-mediated cell death. That both polymerization and depolymerization disrupt the process suggests that active reorganization of the actin network is a required step for the efficient induction of cell death by CD47. Cofilin plays an essential role in actin dynamics by severing F-actin to promote its depolymerization and turnover [ 11 ]. Recently, Csányi et al. reported that ligation of CD47 by thrombospondin increased cofilin activation during micropinocytosis in macrophages [ 42 ]. Our results similarly show that cofilin activity increases upon CC2C6 treatment and is associated with higher sensitivity; the hypersensitive Jkt75 cells had significantly more activated cofilin compared to Jkt25. These data suggest a role for cofilin downstream of PAKs in regulating CD47-mediated cell death. Furthermore, because the ARP2/3 complex nucleates branched actin following cofilin-mediated severing, and WAS patients—who have impaired expression of the ARP2/3 regulator WASP—are insensitive to CD47-mediated cell death [ 6 ], it is likely that this death pathway requires branched actin formation. CD47 clustering and mobility. In mature erythrocytes, CD47 is stabilized on the membrane surface by its inclusion in the band 3 macrocomplex, which links to actin via protein 4.2. However, immature erythrocytes, which lack protein 4.2, rely on the actin cytoskeleton for stability; in those cells, CytoD was found to decrease cell surface CD47 [ 14 ]. In contrast, CD47 remained well expressed in our T-leukemic cell models in the presence of CytoD. Therefore, the decrease in CC2C6 activity following CytoD treatment cannot be explained by the loss of surface CD47. Instead, CD47 has been shown to cluster during cell death and antibody cross-linking, the latter of which induces cytoskeletal rearrangements [ 12 , 43 , 44 ]. Thus, CytoD may disrupt the clustering of CD47 required for downstream cell death signaling. This parallels Fas/CD95, which also clusters during UV- or CD95-mediated apoptosis, though actin disruption did not inhibit death in some models [ 45 , 46 ]. However, consistent with our findings, Parlato et al. found that CytoD inhibited the polarization and uropod formation associated with CD95-mediated death in T cells and in the T-ALL CEM cell line [ 47 ]. While CD95-mediated death is the prototypic example of apoptosis, both Fas and CD47 can induce caspase-independent phenotypes requiring RIPK [ 46 , 48 ]. Additionally, ezrin (an ERM family member) links CD95 to the cytoskeleton (Lozupone, Lugini et al. 2004, Hebert, Potin et al. 2008, Wajant 2014), while radixin was recently shown to directly associate with CD47 in several cancer types [ 49 , 50 , 51 ]. It will be of interest to determine if CD47-mediated death depends on ERM family members or RIPK as a mediator. Cell aggregation and mobility. Dufour et al. recently found that CD47 mobility increased during apoptosis and following integrin activation [ 52 ]. This mobility was highly dependent on cholesterol-containing lipid rafts; disrupting these rafts prevented CD47 clustering, actin rearrangements, cell spreading and SIRPα binding [ 12 , 18 , 53 , 54 ]. In light of our results, it will be interesting to determine if and how CC2C6-mediated cell death affects CD47 mobility and what effects CytoD might have on this. We previously reported that CD47 ligation induces cell aggregation [ 7 ]. Here, we show that preventing this aggregation inhibits cell death, and furthermore, CD47-mediated aggregation can be inhibited by CytoD, suggesting aggregation occurs downstream of actin remodeling. Ligation of CD47 has also been linked to the phosphorylation of vascular endothelial cadherin (VEC) and increased permeability, and fibroblast aggregation has been shown to depend on CD47 homophilic interactions [ 12 , 13 ]. Given that CD47 is known to colocalize with E-cadherin, ZO-1, and cortical actin in epithelial cells [ 12 , 16 ], it will be interesting to determine if CD47-mediated death requires specific cell-cell adhesion structures. Conclusion This paper describes PAKs as new regulators of CD47-mediated cell death. Our data show that PAK signaling can be modulated to synergize with CD47 ligation by way of the actin cytoskeleton. Furthermore, we demonstrate that actin-dependent cell aggregation is a required step for the efficient induction of CD47-mediated cell death. Methods and Materials Patient-derived leukemia xenografts Studies involving human participants were conducted in accordance with the Declaration of Helsinki. Collection and use of patient-derived leukemia cells were approved by the University of British Columbia (UBC) and Children’s & Women’s Health Centre of British Columbia Research Ethics Board (REB) under protocol H14-02930 and H17-01860. Written informed consent was obtained from all participants or their legal guardians prior to sample collection and deposition into the BC Children’s Hospital (BCCH) Biobank. All samples were de-identified to ensure patient confidentiality before use in research. All animal experiments were performed in accordance with the ethical guidelines of the Canadian Council on Animal Care (CCAC). Experimental protocols were reviewed and approved by the University of British Columbia (UBC) Animal Care Committee under protocol A19-0197. Cells from patient-derived xenografts were obtained by injecting NOD scid gamma (NSG) mice (Jackson Laboratory) with whole bone marrow mononuclear cells from patients and viably cryopreserving splenic cells from moribund mice. Cells used were from patients designated BT09, BT012, BT007 (T-ALLs) and BB007, BB013, BB026, BB031, BB039, BB050 (B-ALLs). For experiments, cells were thawed out and allowed to recover overnight or up to 4 days in StemSpan SFEMII with CC100 for B-ALL cells, or in StemSpan SFEMII with Immunocult Human CD3/CD28/CD2 T cell Activator and 100ng/mL IL-2 for T-ALL cells (StemCell Technologies). Cells and tissue culture The following leukemic cell lines were used in this study: Jurkat T-ALL clone E6-1 (ATCC TIB-152); MOLT-4 T-ALL (ATCC CRL-1582), NALM6 B-ALL clone G5 (ATCC CRL-3273), and BV-173 chronic myeloid leukemia (DSMZ ACC 20). Jkt75 is a non-clonal natural derivative of Jurkat E6-1 (ATCC TIB-152, here also referred to as Jkt25), that through routine monitoring was found to exhibit increased CC2C6-induced cell death. Short tandem repeat (STR) analysis confirmed both Jkt75 and Jkt25 with STR profile matching ATCC TIB-152. All cells were maintained in complete RPMI (cRPMI, which is RPMI 1640 (Sigma-Aldrich) with 10% fetal bovine serum (Invitrogen), penicillin/streptomycin (Gibco), and non-essential amino acids (Invitrogen)) at 37°C and 5% CO 2 . Antibodies and small molecule inhibitors CD47 antibodies used were clones CC2C6 (Biolegend) and B6H12 (BD Biosciences). Western blotting antibodies used were as follows: PAK1 (#2602), phospho-PAK1 (Thr423)/PAK2 (Thr402) (#2601), cofilin (#5175), and phospho-cofilin (#3313) were from CST; PAK4 (sc-393367) and phospho-PAK4 (Ser474)(sc-135775) were from Santa Cruz Biotechology; and GAPDH was from Biolegend. Secondary antibodies were goat anti-mouse DyLight488, AlexaFluor 633 or AlexaFluor plus 800, goat anti-rabbit Dylight680 or AlexaFluor plus 800, and goat anti-rat Dylight680. The pan-PAK (PF-3758309), PAK-1 (NVS-PANK-1), PAK-4 (KPT9274), CDC42 (ML-141), Rac1 (EHop-016), and PKD (CID755673) inhibitors were from Selleckchem. Additional inhibitors included Y-27632 (StemCell Technologies), cytochalasin D (Tocris or Selleckchem), and jasplakinolide (Santa Cruz Biotechnology). All were dissolved in DMSO (Sigma-Aldrich) as per manufacturer recommendations. Cell death assays and F-actin labelling Cells were harvested by centrifugation, resuspended in cRPMI at 6x10 5 cells/mL, and incubated in a 12-well plate (Thermo) at 37°C with or without 250ng/mL or 500ng/mL CC2C6 for 2 hours or 24 hours. As indicated, cells may also be co-incubated with inhibitors or with DMSO vehicle control. Cells were then transferred to FACS tubes and washed in PBS. For cell death assays, samples were resuspended in binding buffer with FITC- or Cy5- conjugated annexin V according to manufacturer instructions (BD Biosciences). In some experiments, cells were incubated in a 1.5mL microcentrifuge tube with or without constant rotation using a MACSmix rotator (Miltenyi Biotec). In others, cells were plated on poly-L-lysine (Sigma)-coated 12-well plates and the cells allowed to settle for 45 minutes at 22 o C before addition of CC2C6 and incubation at 37°C. For F-actin determination, cells were fixed and permeabilized using the BD Cytofix/ Cytoperm plus Fixation and Permeabilization kit (BD Biosciences) as per manufacturer recommendation and stained with phalloidin-FITC (Sigma-Aldrich) or pCofillin. Flow cytometry was performed using an AccuriC6 or C6plus (BD Biosciences) and data analyzed using FlowJo (BD). Proteomics Samples were incubated at 37°C for 30 minutes with or without CC2C6, washed in PBS, split equally and centrifuged once more. Supernatants were removed and samples were snap-frozen in liquid nitrogen and stored at -80°C freezer until extraction for proteomics. Cells were lysed in buffer containing 1% SDS (Fisher BioReagents), and processed with single-pot solid-phase-enhanced sample processing (SP3). Peptide digests were washed on a C18 spin column (Nest Group Inc) with 2% acetonitrile (ACN) in 0.1% formic acid, eluted with 80% acetonitrile in 0.1% formic acid (FA) and dried in a Speed Vac. Dried samples were resuspended in 0.1% TFA in 80% ACN and phosphorylated peptides purified by immobilized metal affinity chromatography (IMAC) using Fe-NTA MagBeads (Cube Biotech). Sample solution was added to beads washed with 0.1% TFA in 80% ACN, and incubated in a thermomixer at 22°C for 30 min. Beads were magnetically isolated to recover the non-phosphorylated peptide fraction in the supernatant. Following two wash steps in sample resuspension buffer, phosphorylated peptides were eluted using 1% ammonia, and immediately acidified to pH 3 with FA. Peptide solutions were cleaned with C18 spin columns (Nest Group Inc) by washing with 0.1% TFA, and eluted with 0.1% FA in 50% ACN. After drying in a Speed Vac, samples were resolubilized in 0.1% FA for mass spectrometry analysis. Liquid chromatography tandem mass spectrometry (LC-MSMS) Data-dependent Acquisition (DDA)—1µg of phosphor-peptides were analyzed on a Q Exactive HF plus Orbitrap mass spectrometer coupled to an Easy-nLC 1200 liquid chromatography (Thermo Scientific) with a 3 cm-long homemade precolumn (Polymicro Technologies capillary tubings, 360OD, 100ID), a 35 cm-long homemade analytical column (Self-pack PicoFrit columns, 360OD, 75ID, 15 m tip ID) and packed with Dr. Maisch beads (ReproSil-Pur 120 C18-AQ, 3 um) with a flow rate at 300 nL/min and constant temperature at 50°C. Mobile phase A (0.1% FA in water) and mobile phase B (0.1% FA in 95% ACN) were used for a 120 min gradient. DDA: A full-scan MS spectrum (350 − 1600 m/z) was collected with resolution of 120,000 at m/z 200 and the maximum acquisition time of 246 ms and an AGC target value of 1e6. MS/MS scan was acquired at a resolution of 60,000 with maximum acquisition time of 118 ms and an AGC target value of 2e5 with an isolation window of 1.4 m/z at Orbitrap cell. The top 12 precursors were selected. Normalized collision energy (NCE) was set to 28. Dynamic exclusion duration was set to 15 s. Charge state exclusion was set to ignore unassigned, 1, and 5 and greater charges. The heated capillary temperature was set to 275°C. Data Processing—Raw MS DDA data acquired on the Q Exactive HF were processed and searched with MaxQuant version 1.6.2.10 using the built-in Andromeda search engine. The first search peptide tolerance of 20 ppm and main search peptide tolerance of 4.5 ppm were used. The human protein database was downloaded from UniProt (release 2018_09; 20,410 sequences) and common contaminants were embedded from MaxQuant. Phosphopeptide and statistical analysis was performed in Perseus and INKA [ 30 ]. SDS-PAGE and western immunoblots Cells were centrifuged, resuspended in cRPMI with or without treatment, and incubated at 37°C for the indicated time. Cells were then washed in PBS and lysed in RIPA buffer (50mM Tris pH8, 150mM NaCl, 1% Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, Complete protease inhibitors (Roche), and 25mM sodium fluoride). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Proteins were labelled with primary and secondary antibodies as indicated and membranes were imaged using the Sapphire FL Biomolecular Imager (Azure Biosystems) or the Odyssey (LICORbio) and analyzed using Image Studio (LICORbio). Microscopy Cells were centrifuged, resuspended in cRPMI with or without treatment in a 12-well plate (Thermo) and incubated at 37°C for 2 hours. Samples were then resuspended by pipetting thrice and cells allowed to settle for 5 minutes before imaging using an Olympus IX81 microscope (4x objective) equipped with a CoolSnap HQ2 camera (Photometrics) and controlled by Metamorph® software (Molecular Devices). Post-acquisition processing was performed on ImageJ. Declarations Conflict of interest The authors declare no conflict of interest. Acknowledgements We extend our sincere gratitude to the patients and families for their invaluable contribution of tissue samples to the BC Children’s Hospital (BCCH) Biobank. This research was made possible by their generous participation and the expert assistance of the biobank staff in sample procurement. Arnawaz Bashir provided technical assistance at various phases of this project. Support from the BCCH Foundation for the Michael Cuccione Childhood Cancer Research Program and the BRAvE initiative enabled portions of the PDX work. This work was supported by the Canadian Institutes of Health Research (CIHR) Project Grant PJT-175116 awarded to C.J.L. Author Contributions P.L. and C.J.L. performed study concept and design, development of methodology and writing, review and revision of the paper; P.L., A.C.U., A.L., P.F.L. and C.J.L. performed acquisition, analysis and interpretation of data, and statistical analysis; N.R., C.A.M. and G.S.D. provided technical and material support. All authors approved the final paper. References Soto-Pantoja DR, Kaur S, Roberts DD. CD47 signaling pathways controlling cellular differentiation and responses to stress. Crit Rev Biochem Mol Biol 2015, 50(3): 212–230. Leclair P, Lim CJ. CD47 (Cluster of differentiation 47): an anti-phagocytic receptor with a multitude of signaling functions. Anim Cells Syst (Seoul) 2020, 24(5): 243–252. 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Additional Declarations There is no duality of interest Supplementary Files SuppFig1.jpg Supplemental Figure 1. Chemosensitivity profiling of BB026-Dx and -R1 to doxorubicin and vincristine. PDX cells were treated with doxorubicin or vincristine for 48hr and cell viability assessed with CellTiter-Glo assay. IC50s were calculated with Prism Graphpad. SuppFig2.jpg Supplemental Figure 2. Phospho-proteomics data from BB026 Dx and R1 samples treated with or without CC2C6 for 30 were subjected to INKA analysis as described in ref. [30]. Proteins with increased kinase activity following CC2C6 treatment are denoted in red, while those with decreased activity are denoted in blue. SuppFig3.jpg Supplemental Figure 3. Synergistic effects of CC2C6 and PAK inhibitor. Cells were incubated with or without CC2C6 and with or without PF-3758309 for 24h as follows: (A-D) Jurkat at 6μM PF, MOLT-4 at 6μM PF, BV-173 at 2μM PF and Nalm6 at 350nM PF. Cell death was assessed by flow cytometry with Annexin V assay. Round dot shown represent the % cell death for an ‘additive-only’ effect between CC2C6 and PF-3758309. Error bars represent SD from ≥3 independent experiments. Significance: ***p<0.001, ****p<0.0001. SuppFig4.jpg Supplemental Figure 4. Phospho-proteomic data from Jkt25 and Jkt75 cells untreated or treated with CC2C6 for 30 minutes were subjected to INKA analysis. SuppFig5.jpg Supplemental Figure 5. CC2C6 treatment on pS3-cofilin in Jkt25 and Jkt75 cells. Cells were treated with CC2C6 for the indicated times and pS3-cofilin assessed by flow cytometry as indicated in methods and materials. SuppFig6.jpg Supplemental Figure 6. (A) Jkt25 and Jkt75 cells were treated with CC2C6 or cytochalasin D at various concentrations for 2 hr and F-actin was assessed by flow cytometry following phalloidin-FITC staining. Shown is the geometric mean of fluorescence (gMFI). (B) Jkt25 and Jkt75 cells were treated with or without cytochalasin D for 2 hr and then stained with anti-CD47 clone B6H12 to assess cell surface CD47 expression by flow. SuppFig7.jpg Supplemental Figure 7. Jkt25, Jkt75, and CD47 -/- cells were treated with or without CC2C6 and cytochalasin D (10μM) for 2 hr and imaged by microscopy. Shown are representative images of three separate experiments. Images were analyzed for cluster size using ImageJ, with clusters pseudocolored as red highlights. Originaldatafiles.pdf Original Data Files Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 22 Apr, 2026 Review # 2 received at journal 19 Apr, 2026 Reviewer # 2 agreed at journal 10 Apr, 2026 Review # 1 received at journal 08 Mar, 2026 Reviewer # 1 agreed at journal 03 Mar, 2026 Reviewers invited by journal 26 Feb, 2026 Submission checks completed at journal 24 Feb, 2026 First submitted to journal 23 Feb, 2026 Unknown event 23 Feb, 2026 Editor assigned by journal 22 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8940785","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":596422800,"identity":"15674547-3993-41ab-b19a-3d0d835c6bb3","order_by":0,"name":"Chinten Lim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYFCCww0HGCoYGNjYGRiYidRyEKjlDFALM/FaGBsYGNsYwOqJ08LfeLDx0M152+T5mBnYpAtqGOT5GwhokThwsOFw7rbbhm0gLTOOMRjOOEBAiwEDRAsjWAtvA0MCA3Fa5ty2h2uRJ05Lw+1EuBYDQlrAfsk5dju5jZmx2XrGMQnDjYS08M84fPhzTs1t2/ntzQdvF9TYyMsR0gK0BsYCRhCQS0g9yJoGIhSNglEwCkbByAYAIC1ALPVqpLMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6381-7585","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Chinten","middleName":"","lastName":"Lim","suffix":""},{"id":596422801,"identity":"22693d7d-1335-41b6-b950-a467701c23ff","order_by":1,"name":"Pascal Leclair","email":"","orcid":"https://orcid.org/0000-0002-0791-7028","institution":"University of British Columbia","correspondingAuthor":false,"prefix":"","firstName":"Pascal","middleName":"","lastName":"Leclair","suffix":""},{"id":596422802,"identity":"81402f30-667c-480a-a82e-5050d00d01bb","order_by":2,"name":"Anuli Uzozie","email":"","orcid":"","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Anuli","middleName":"","lastName":"Uzozie","suffix":""},{"id":596422803,"identity":"abd7e326-60fa-4504-9c70-2e661fd6d5f4","order_by":3,"name":"Amanda Lorentzian","email":"","orcid":"","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"","lastName":"Lorentzian","suffix":""},{"id":596422804,"identity":"0213fd0b-ee51-4b12-8c1e-aa512c3d34b0","order_by":4,"name":"Nina Rolf","email":"","orcid":"","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Rolf","suffix":""},{"id":596422805,"identity":"7ffd38ad-e226-434e-9f71-9782b6406143","order_by":5,"name":"Christopher Maxwell","email":"","orcid":"","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Maxwell","suffix":""},{"id":596422806,"identity":"bc8f6a36-0600-451a-b64a-f0fe73d16502","order_by":6,"name":"Gregor Reid","email":"","orcid":"","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Gregor","middleName":"","lastName":"Reid","suffix":""},{"id":596422807,"identity":"0657dab0-b707-422c-8d55-9095360d60e8","order_by":7,"name":"Philipp Lange","email":"","orcid":"","institution":"University of British Columbia / BC Children's Hospital Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Lange","suffix":""}],"badges":[],"createdAt":"2026-02-22 18:05:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8940785/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8940785/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104408977,"identity":"dee6af77-9c92-4475-ad53-99fbffe6d589","added_by":"auto","created_at":"2026-03-11 12:43:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1395002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD47 ligation mediated cell death in T-ALL and B-ALL PDX models and cell lines.\u003c/strong\u003e (\u003cstrong\u003eA–C\u003c/strong\u003e) Cell death assessment via Annexin V flow cytometry following 2 hours of CC2C6 treatment. Data represent (\u003cstrong\u003eA\u003c/strong\u003e) T-ALL PDXs compared to Jurkat cell line and (\u003cstrong\u003eB, C\u003c/strong\u003e) B-ALL PDXs compared to BV-173 cell line. (\u003cstrong\u003eD\u003c/strong\u003e) Evaluation of CD47 expression in PDX samples (BB0026 Dx and R1) relative to Jurkat cells, using anti-CD47 clones B6H12 and CC2C6. Error bars represent SD of technical triplicates (\u003cstrong\u003eA\u003c/strong\u003e) or biological replicates from four independent experiments (n=4) (\u003cstrong\u003eC\u003c/strong\u003e). Significance: *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/c845b13fc403947aef5ecb4d.png"},{"id":104409279,"identity":"a3855a66-4416-4f63-939a-bfd0af00e3c0","added_by":"auto","created_at":"2026-03-11 12:44:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4128096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhospho-proteomic profiling reveals differential signaling following CC2C6-CD47 engagement.\u003c/strong\u003e Volcano plots display phospho-enriched cell lysates from (\u003cstrong\u003eA\u003c/strong\u003e) BB026-Dx and (\u003cstrong\u003eB\u003c/strong\u003e) BB026-R1 PDX samples treated with CC2C6 for 30 minutes vs. untreated controls (n=3 per group). Phospho-proteins significantly enriched after CC2C6 treatment are highlighted in red, while those significantly enriched in untreated samples are highlighted in blue.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/6429e4cdabd86382977372d7.png"},{"id":104408984,"identity":"e4b5d9d4-badd-40fe-8a51-c640430b1915","added_by":"auto","created_at":"2026-03-11 12:43:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2688467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic induction of cell death by PAK inhibition and CC2C6 treatment.\u003c/strong\u003e BB026-Dx and R1 cells were treated with CC2C6 in combination with the pan-PAK inhibitor PF-3758309. Cell death was assessed following treatment at: (\u003cstrong\u003eA\u003c/strong\u003e) 5μM PF for 2hr, and (\u003cstrong\u003eB\u003c/strong\u003e) 175nM PF for 24hr. Cell death was assayed for the following cells co-treated with CC2C6 and PF-3758309: (\u003cstrong\u003eC\u003c/strong\u003e) Jurkat at 200nM PF for 2hr, or 50nM for 24hr; (\u003cstrong\u003eD\u003c/strong\u003e) MOLT-4 at 1.5μM PF for 2hr, or 500nM for 24hr; (\u003cstrong\u003eE\u003c/strong\u003e) BV-173 at 2.5μM PF for 2hr, or 500nM for 24hr; (\u003cstrong\u003eF\u003c/strong\u003e) Nalm6 at 500nM PF for 2hr, or 50nM for 24hr. Jurkat cells were treated with CC2C6 and (\u003cstrong\u003eG\u003c/strong\u003e) PAK1/2/3 inhibitor (NVS-PAK1-1) at 1μM for 2hr, or 15μM for 24hr, or, (\u003cstrong\u003eH\u003c/strong\u003e) PAK4 inhibitor (KPT9274) at 2.5μM for 2hr, or 10μM for 24hr. Cell death was quantified via Annexin V flow cytometry assay. Round dot shown for dual treatments represent the % cell death for an ‘additive-only’ effect, calculated as: % death with CC2C6 alone + % death with inhibitor alone (eg PF) - % death for untreated. Error bars represent SD from ≥3 independent experiments. Significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/c6d65dfc2eef95d21eeca6a1.png"},{"id":104409469,"identity":"73e9548c-342f-4893-a0fa-8eb20f84e226","added_by":"auto","created_at":"2026-03-11 12:45:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1425288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynergistic induction of cell death by combined CC2C6 treatment with Rho, PKD or ROCK inhibition. \u003c/strong\u003eCell death was assessed for Jurkat cells co-treated with CC2C6 and (\u003cstrong\u003eA\u003c/strong\u003e) CDC42 inhibitor (ML-141) at 20μM for 2hr, or 5μM for 24hr; (\u003cstrong\u003eB\u003c/strong\u003e) Rac1 inhibitor (EHop-016) at 25μM for 2hr, or 5μM for 24hr; (\u003cstrong\u003eC\u003c/strong\u003e) PKD inhibitor (CID755673) at 50μM for 2hr, or 15μM for 24hr; (\u003cstrong\u003eD\u003c/strong\u003e) ROCK inhibitor (Y-27632) at 100μM for 2hr, or 10μM for 24hr. Round dot shown represent the % cell death for an ‘additive-only’ effect. Error bars represent SD from ≥3 independent experiments. Significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/96446d8d922f284715912d12.png"},{"id":104408890,"identity":"42cf596e-7771-4afc-9821-dff679674261","added_by":"auto","created_at":"2026-03-11 12:43:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3526454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation of CC2C6 induced cell death with PAK and cofilin signaling.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Cell death was assessed for Jkt25 and Jkt75 cells treated with or without CC2C6 for 2hr. Cells were treated with CC2C6 for 30min or 2hr and lysates analyzed by western immunoblots for the indicated total and phospho-proteins. (\u003cstrong\u003eB\u003c/strong\u003e) Shown is a representative experiment from at least three separate experiments. (\u003cstrong\u003eC-E\u003c/strong\u003e) Densitometric quantification of western blot data. Band intensities were normalized to GAPDH and to the Jkt25 untreated samples. (\u003cstrong\u003eF\u003c/strong\u003e) Jkt25 cells were treated with CC2C6 for the indicated times and pS3-cofilin expression assessed by flow cytometry. Error bars represent SD from 3 independent experiments (\u003cstrong\u003eC-F\u003c/strong\u003e). Significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/d3e7513a79fbf7a821a842e2.png"},{"id":104408904,"identity":"f05ddb08-f0d9-47f0-ac05-579f41db6d07","added_by":"auto","created_at":"2026-03-11 12:43:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2796441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eF-actin cytoskeleton modulates CD47-mediated cell death.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Jkt25 and Jkt75 cells, or (\u003cstrong\u003eB\u003c/strong\u003e) PDX B0026 Dx and R1 cells, were treated with 2.5μM cytochalasin D (CytoD) for 4hr or CC2C6 for the indicated times, fixed, and F-actin stained with phalloidin-FITC. Flow cytometry geometric mean of fluorescence intensity (gMFI) is reported. % F-actin was calculated as: (gMFI treatment/gMFI untreated) *100. Error bars represent SD from 3 independent experiments. (\u003cstrong\u003eC-F\u003c/strong\u003e) Cell death was assessed for the indicated cells treated with or without CC2C6, and, with or without 10μM cytochalasin D for 2hr. In addition, cells also received combination treatment with 5μM PF-3758309 (\u003cstrong\u003eG, H\u003c/strong\u003e) or 2μM jasplakinolide (\u003cstrong\u003eI\u003c/strong\u003e). Round dot shown represent the % cell death for an ‘additive-only’ effect between CC2C6 and PF-3758309 (\u003cstrong\u003eG,H\u003c/strong\u003e). Error bars represent SD from 3 independent experiments. Significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/b02aa6ffa923f194e5dac50c.png"},{"id":104408983,"identity":"355228e9-6d23-43e3-bc99-f9e175adfc15","added_by":"auto","created_at":"2026-03-11 12:43:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1421210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytochalasin D disrupts CC2C6-mediated cell aggregation.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Jkt25, (\u003cstrong\u003eB\u003c/strong\u003e) Jkt75, and (\u003cstrong\u003eC\u003c/strong\u003e) CD47\u003csup\u003e-/-\u003c/sup\u003e cells were treated with or without CC2C6 and cytochalasin D (10μM) for 2 hr and imaged by microscopy. The images were analyzed for cluster size using ImageJ and binned as indicated. Error bars represent SD from 3 independent experiments, using 5 fields of view for each condition analyzed from each experiment. Significance: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/8b63f0e8aa809b8097f3c59a.png"},{"id":104408907,"identity":"421f854c-f969-49af-84b3-e821f78c868c","added_by":"auto","created_at":"2026-03-11 12:43:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":89464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell-cell contacts facilitate efficient CD47-mediated cell death. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Jkt25 and Jkt75 cells were treated for 2hr with CC2C6 in microcentrifuge tubes with or without constant rotation. (\u003cstrong\u003eB\u003c/strong\u003e) Jkt25 and Jkt75 cells were seeded in 12-well multidish, without or with poly-L-lysine (PLL) coating. Cell death was assessed using Annexin V flow cytometry. Error bars represent SD from 3 independent experiments. Significance: ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/e54d868f16b45c9ab3ac0b0d.png"},{"id":104415897,"identity":"e7e8891b-0074-4dbf-8ed4-cfc093ae0109","added_by":"auto","created_at":"2026-03-11 13:12:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20653610,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/23b077e9-5981-40bb-80a9-630e7795e470.pdf"},{"id":104408865,"identity":"47865925-6342-41a5-a3e8-05e2abf95791","added_by":"auto","created_at":"2026-03-11 12:43:36","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":830850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 1.\u003c/em\u003e Chemosensitivity profiling of BB026-Dx and -R1 to doxorubicin and vincristine. PDX cells were treated with doxorubicin or vincristine for 48hr and cell viability assessed with CellTiter-Glo assay. IC50s were calculated with Prism Graphpad.\u003c/p\u003e","description":"","filename":"SuppFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/580827226c7c0f4466b85ff2.jpg"},{"id":104409106,"identity":"8cbfb150-f867-45d9-b660-2014b82e1fba","added_by":"auto","created_at":"2026-03-11 12:44:08","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3293626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 2.\u003c/em\u003e Phospho-proteomics data from BB026 Dx and R1 samples treated with or without CC2C6 for 30 were subjected to INKA analysis as described in ref. [30]. Proteins with increased kinase activity following CC2C6 treatment are denoted in red, while those with decreased activity are denoted in blue.\u003c/p\u003e","description":"","filename":"SuppFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/3ffb4c7fff0359f53630d1ac.jpg"},{"id":104408883,"identity":"de3d9e2e-a89b-4487-99cb-362553cc2aad","added_by":"auto","created_at":"2026-03-11 12:43:39","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":610492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 3.\u003c/em\u003e Synergistic effects of CC2C6 and PAK inhibitor. Cells were incubated with or without CC2C6 and with or without PF-3758309 for 24h as follows: (\u003cstrong\u003eA-D\u003c/strong\u003e) Jurkat at 6μM PF, MOLT-4 at 6μM PF, BV-173 at 2μM PF and Nalm6 at 350nM PF. Cell death was assessed by flow cytometry with Annexin V assay. Round dot shown represent the % cell death for an ‘additive-only’ effect between CC2C6 and PF-3758309. Error bars represent SD from ≥3 independent experiments. Significance: ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"SuppFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/37e3d00a88652c514f08ee5f.jpg"},{"id":104408622,"identity":"1ee6df5d-683f-49a4-81ea-c91b5f563b52","added_by":"auto","created_at":"2026-03-11 12:42:57","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3951336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 4.\u003c/em\u003e Phospho-proteomic data from Jkt25 and Jkt75 cells untreated or treated with CC2C6 for 30 minutes were subjected to INKA analysis.\u003c/p\u003e","description":"","filename":"SuppFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/5cd1c1ea0dd7988067a89ef4.jpg"},{"id":104412817,"identity":"2d695d5f-0e96-4e1b-8eef-a2eb886c8a69","added_by":"auto","created_at":"2026-03-11 13:01:15","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":623821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 5.\u003c/em\u003e CC2C6 treatment on pS3-cofilin in Jkt25 and Jkt75 cells. Cells were treated with CC2C6 for the indicated times and pS3-cofilin assessed by flow cytometry as indicated in methods and materials.\u003c/p\u003e","description":"","filename":"SuppFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/dd425b4d5943715b871da7de.jpg"},{"id":104408624,"identity":"bbf7a714-7feb-42dc-b0ac-8683edb438db","added_by":"auto","created_at":"2026-03-11 12:42:58","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":614763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 6. \u003c/em\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Jkt25 and Jkt75 cells were treated with CC2C6 or cytochalasin D at various concentrations for 2 hr and F-actin was assessed by flow cytometry following phalloidin-FITC staining. Shown is the geometric mean of fluorescence (gMFI). (\u003cstrong\u003eB\u003c/strong\u003e) Jkt25 and Jkt75 cells were treated with or without cytochalasin D for 2 hr and then stained with anti-CD47 clone B6H12 to assess cell surface CD47 expression by flow.\u003c/p\u003e","description":"","filename":"SuppFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/5012ec1485a6c5bd01225c69.jpg"},{"id":104409347,"identity":"c6981c34-965d-43f8-8af0-6a4215c5c543","added_by":"auto","created_at":"2026-03-11 12:44:49","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12924711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSupplemental Figure 7. \u003c/em\u003eJkt25, Jkt75, and CD47\u003csup\u003e-/-\u003c/sup\u003e cells were treated with or without CC2C6 and cytochalasin D (10μM) for 2 hr and imaged by microscopy. Shown are representative images of three separate experiments. Images were analyzed for cluster size using ImageJ, with clusters pseudocolored as red highlights.\u003c/p\u003e","description":"","filename":"SuppFig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/f29932ff50663d269823ece5.jpg"},{"id":104408814,"identity":"b9df4d91-bf8c-4088-8dc0-09240e3348ac","added_by":"auto","created_at":"2026-03-11 12:43:30","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":321160,"visible":true,"origin":"","legend":"Original Data Files","description":"","filename":"Originaldatafiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8940785/v1/e9c0a4151e15d93319259fde.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"\u003cp\u003eRegulation of CD47-mediated cell death by p21-activated kinases\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCD47 is a plasma membrane penta-spanning cell-surface glycoprotein best known as a \u0026lsquo;don\u0026rsquo;t eat\u0026rsquo; anti-phagocytic signal upon engaging SIRPα, its counter receptor expressed on professional phagocytes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Via its association with integrins, CD47 is also involved in cell adhesion and migration. However, a lesser studied role for CD47 is its ability to induce cell death in a variety of cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Early investigations into CD47 revealed its role in inducing a rapid onset of cell death when ligated by antibodies or its natural ligand, thrombospondin (TSP). The features of CD47-mediated cell death include phosphatidylserine (PS) exposure and mitochondrial abnormalities, such as ROS production and decreased mitochondrial membrane potential. However, in contrast to classical apoptosis, CD47-mediated cell death (at one time coined as \u0026lsquo;type III\u0026rsquo;) is caspase-independent, does not involve cytochrome \u003cem\u003ec\u003c/em\u003e release from mitochondria, nor does it involve nuclear alterations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. We reported previously the CD47 antibody clone CC2C6 efficiently induced cell death in solution, in contrast to clone B6H12 which requires immobilization [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrior studies have identified a role for actin dynamics in modulating CD47-mediated cell death, as exemplified by the inhibitory effects of the actin depolymerizer, cytochalasin D [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, Mateo \u003cem\u003eet al.\u003c/em\u003e found that cells isolated from Wiskott-Aldrich syndrome (WAS) patients, who have abnormal expression of WAS protein (WASp, an actin nucleation-promoting factor of the Arp2/3 complex) were insensitive to CD47-mediated cell death [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Microscopy based studies also showed alterations in filamentous actin following CD47 ligation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Actin involvement in CD47 processes is not limited to cell death but has also been shown to increase CD47 homophilic cell aggregation in fibroblasts [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], stabilize CD47 at the cell membrane of erythrocytes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and induce the formation of stress fibers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] or cortical bundles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, CD47 colocalized with cortical actin, E-cadherin, and ZO-1 at cell-cell-contacts of epithelial cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and with fascin, an actin bundling protein, at the tips of microspikes generated by COMP (\u003cem\u003ei.e.\u003c/em\u003e, TSP-5) binding to CD47 in ligament cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Finally, CD47 ligation has been shown to induce the depolymerization and degradation of actin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while others have shown an increase in F-actin following CD47 ligation, a phenomenon that was amplified by CD3 ligation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eActin dynamics are regulated by G proteins via a variety of intermediaries, such as p21-activated kinases (PAKs), cofilin, Arp2/3, and WASp [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The PAK family of proteins are distinguished by their regulatory domain and mechanism of activation. Binding of activated small GTPases to group I PAKs (PAK1-3) induce a conformational change that enables their phosphorylation and activation. In contrast, group II PAKs (PAK4-6) are constitutively phosphorylated at the equivalent site and their activation seems to be determined solely on the binding of small GTPases or an SH3 domain, though the precise mechanism is still under investigation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Nonetheless, activation of PAKs leads to phosphorylation and activation of LIMK, which in turn leads to phosphorylation and inactivation of cofilin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cofilin regulates actin dynamics by binding and severing F-actin, which initiates actin re-assembly into various structures depending on the different proteins present. For example, WASp and the Arp2/3 complex are involved in building branched networks in lamellipodia, whereas fascin aids in forming parallel bundles in filopodia [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, the actin cytoskeleton is also important in establishing and maintaining various types of cell-cell junctions, including immunological synapses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, consisting of precursor B and T subtypes (B-ALL and T-ALL) occurring at a 17:3 ratio. Although the 5-year survival rate for childhood leukemia now approaches 90%, up to 20% of patients relapse at least once and require treatment intensification [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Importantly, the highly toxic drugs administered to children undergoing chemotherapy at any stage result in serious long-term sequelae, including developmental defects, cardiotoxicities, neurotoxicities, and secondary malignancies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As such, the development of novel adjuvant or second-line therapies that enable reduced dosing of conventional cytotoxic agents continue to be needed [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we extend the findings of the actin cytoskeleton as a downstream regulator of CD47-mediated cell death in ALL and show that a synergistic increase in CC2C6-mediated cell death can be achieved by inhibition of PAKs upstream of cytoskeleton dynamics. Furthermore, we show that a derivative Jurkat cell line exhibits very high levels of CD47-mediated cell death that is correlated with reduced activated PAKs and increased F-actin compared to its isogenic parental strain. Finally, we show that actin-mediated cell aggregation is required for maximal sensitivity to CC2C6.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePAK signaling regulates CD47-mediated cell death.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously, we showed that treating Jurkat T-ALL cells with CC2C6 induces significant cell death in as little as 30-minutes, with maximal effect observed between 2\u0026ndash;6 hours [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, this response is not consistent across a number of ALL cell lines tested, including in patient-derived xenografts (PDX) of B- and T-ALLs. For example, in T-ALLs, BT009 showed no significant response and BT012 only a marginal one (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Of note, multiple PDXs of BT007, which included leukemic blasts from diagnosis (Dx), at end-of-consolidation (D29) and at relapse (R1), exhibited comparable and conserved response to CC2C6 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In B-ALLs, BB007, BB013, BB039 and BB050 showed minimal to no response to CC2C6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Of particular significance, the relapsed tumor BB026-R1 exhibited almost a two-fold increase in CC2C6-induced cell death when compared to the diagnostic tumor BB026-Dx (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Relapsed leukemias typically exhibit increased resistance to standard treatments like doxorubicin and vincristine, a phenomenon also observed for BB026-R1 when compared to BB026-Dx (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The levels of cell surface expressed CD47 was comparable between BB026-Dx and -R1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), suggesting the difference in CC2C6-induced cell death is likely due to differences in cell intrinsic signaling unrelated to CD47 levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRationalizing that differences in kinase activities may underlie the differential sensitivity of BB026-Dx and -R1 cells to CD47-ligation, we performed phospho-proteome analysis of cells without and with CC2C6 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Using Inferred Kinase Activity (INKA) analysis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], we noted a number of kinases with actin regulatory functions were downregulated following CD47-ligation, including ROCK1, ROCK2, PAK1, PAK2 and PAK4 (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To understand the role of PAKs in CD47-mediated cell death, we treated cells with CC2C6 and the pan-PAK inhibitor, PF-3758309 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Co-treatment of CC2C6 and PF-3758309 for 2 hrs resulted in increased cell death occurring in a synergistic manner for BB026-Dx, but not for BB026-R1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Consistent with our prior findings [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], treatment with CC2C6 for 24 hrs resulted in lower levels of cell death compared to 2 hrs, but also facilitated longer incubation with a secondary agent. In this manner, co-treatment of CC2C6 and PF-3758309 for 24 hrs resulted in increased and synergistic cell death observed for both BB026-Dx and -R1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that PAK activity regulates CD47-ligation mediated cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then assessed if this phenomenon could be more widely replicated in various leukemic cell lines. Synergistic activities were observed for Jurkat, MOLT-4 and BV-173 cells treated with CC2C6 and PF-3758309 for either 2 or 24 hrs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). Our testing was also extended to NALM6 cells, a B-ALL cell line that exhibited minimal CD47-ligation induced cell death in our standard 2 hr assay. At best, combined CC2C6 and PF-3758309 treatment for 2 hrs revealed only a small and additive effect, while a 24 hrs treatment revealed a clear synergistic effect on cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Whenever possible, we selected a monoagent concentration of PF-3758309 that yielded a minimal effect on cell death for each assay condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). It is notable that the observed synergistic effects remained evident for cells treated with CC2C6 and a higher concentration of PF-3758309 (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePF-3758309 is a potent inhibitor of group I and group II PAKs, including PAK1 and PAK4. To assess if CD47-mediated cell death may involve differential regulation by group I or II PAKs, we also tested the effect of PAK1 or PAK4 inhibition using the selective inhibitors, NVS-PAK1-1 and KPT9274 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In Jurkat cells, CC2C6 had much lower synergistic effects with PAK1- or PAK4- selective inhibitors when compared with the pan-PAK inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H), suggesting that this is not an isoform-specific phenomenon.\u003c/p\u003e \u003cp\u003eNext, we assessed the role of proteins known to regulate PAK activity, including the Rho-family of small GTPases and protein kinase D (PKD)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the latter of which was also implicated in our INKA analysis (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The Cdc42 inhibitor, ML-141 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], exhibited modest synergy with CC2C6 to induce Jurkat cell death after 2 and 24 hr treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, synergistic activity was only observed following 24 hr co-treatment with the Rac1 inhibitor, EHop-016 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). As for the pan PKD inhibitor, CID755673 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], synergistic activity was only observed with 2 hr co-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Finally, we assessed the inhibition of the actin regulator, ROCK, since it was implicated in our INKA analysis and its function parallels that of PAK. As such we found synergy between the ROCK inhibitor, Y-27632 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and CC2C6 at both 2 and 24 hr incubations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Taken together, our results reveal that CD47 ligation-induced cell death is enhanced by inhibition of PAK signaling and regulators of actin cytoskeletal remodeling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression and activity of PAK and cofilin in CD47-mediated cell death.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSerendipitously, we derived a substrain of Jurkat cells, termed Jkt75, which consistently exhibited much greater levels of CC2C6-induced death, up to 75%, when compared to the ~\u0026thinsp;25% observed in the parental cells (herein termed Jkt25, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar to our results using BB026 cells, INKA analysis of phospho-proteomic data of Jkt25 and Jkt75 cells following CC2C6 treatment revealed similar downregulated activity of PAKs (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To understand the role of PAKs in CD47-mediated cell death, we compared the expression of phosphorylated and total PAK1 and PAK4 with and without CC2C6 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Compared to Jkt25, Jkt75 cells expressed higher levels of PAK1, and lower levels of pT423-PAK1 and pS474-PAK4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). CD47 ligation increased the expression of PAK1 in Jkt25 cells, but did not result in measurable change of pPAK1 or pPAK4 in either cell types. As a proxy for PAK activation, we also assessed if the downstream actin regulator, cofilin, might be differentially regulated after CC2C6 treatment. While total cofilin levels were comparable in both Jkt25 and Jkt75 cells, the inactive pS3-cofilin was significantly higher in Jkt25 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), consistent with the increased pT423-PAK1 and pS474-PAK4 observed. As the immunoblot-based assays provided limited resolution of changes induced by CC2C6 treatment, we utilized an intracellular flow cytometry-based assay for pS3-cofilin to enhance the temporal resolution of changes associated with actin cytoskeletal dynamics (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). CC2C6 treatment of Jkt25 cells revealed a bi-phasic response in pS3-cofilin, with an initial decrease observed at 15mins and a second phase after 1 hr (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Taken together, these results suggest that increased sensitivity to CD47-mediated cell death is correlated with reduced PAK signaling and dynamic changes in cofilin activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eActin cytoskeletal dynamics modulate CD47-mediated cell death.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that CD47 ligation induces F-actin depolymerization, and that cytochalasin D (CytoD) treatment attenuates CD47-mediated cell death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Given that PAK activity regulates the actin cytoskeleton and we observe increased cofilin activity following CC2C6 treatment, we assessed if CD47-ligation with CC2C6 also modulates the actin cytoskeleton.\u003c/p\u003e \u003cp\u003eCD47-ligation with CC2C6 resulted in a significant decrease in F-actin in both Jkt25 and Jkt75 cells, with the latter exhibiting a more substantive decrease correlating with higher basal F-actin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We observed that CC2C6 treatment resulted in even lower F-actin levels in Jkt75 cells compared to the most potent concentration of CytoD (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), suggesting the involvement of distinct F-actin depolymerization mechanisms in Jkt75 cells. A significant decrease in F-actin levels following CC2C6 treatment was seen for B0026-R1 cells, but not for B0026-Dx cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We found that CytoD significantly attenuated cell death induced by CC2C6 in Jkt25, Jkt75, B0026-Dx, B0026-R1, MOLT4, and BV-173 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F). Cell surface CD47 expression was unchanged with CytoD treatment (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), hence the cell death inhibition observed was not attributed to decreased receptor function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince PAK is a known up-stream regulator of actin dynamics, we tested if CytoD could negate the synergistic effects of combined CD47-ligation and PAK-inhibition. Indeed, cell death decreased by more than half in both Jkt25 and Jkt75 cells when CytoD was incubated along with CC2C6 and PF-3758309 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG,H). We also found that CC2C6-induced cell death was attenuated in cells co-treated with jasplakinolide (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), a binder of actin and F-actin stabilizer [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Given that both CytoD and jasplakinolide share the ability to disrupt actin function, we surmised that CD47-mediated cell death involves dynamic remodeling of the F-actin cytoskeleton.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCD47-mediated cell death requires actin-mediated cell-cell contacts.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe showed previously that CC2C6 treatment led to Jurkat cell-cell aggregation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given that F-actin is involved in reinforcing cell-cell contacts [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], we hypothesized that CD47-mediated cell death involves actin cytoskeletal remodeling and cell-cell aggregation. Indeed, Jkt75 cells exhibited greater CC2C6 treatment-induced aggregation when compared to Jkt25, with CD47\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells showing no response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C, \u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Consistent with the requirement for F-actin cytoskeletal remodeling, co-incubation with CytoD and CC2C6 reduced the formation of aggregates for both Jkt25 and Jkt75 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also assessed if physical limitation of cell-cell contacts is sufficient to inhibit CD47-ligation induced cell death by incubating cells under constant vessel movement (rotation) or pre-immobilized on poly-L-lysine coated dishes. Under these conditions, we found that cell death was significantly inhibited for Jkt25 and Jkt75 cells treated with CC2C6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B). In sum, our cumulative data are consistent with the interpretation that CD47-ligation mediated cell death requires modulation of F-actin dynamics and cell-cell aggregation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003ePAKs as novel regulators of CD47-mediated cell death.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe demonstrate for the first time that members of the p21-activated kinase (PAK) family regulate CD47-mediated cell death via the actin cytoskeleton. Initial phospho-proteomic analysis of PDX B-ALL cells suggested the activity of several PAKs were downregulated following treatment with CD47 antibody (clone CC2C6), which correlated with induction of cell death. This phenomenon was also observed in the Jurkat T-ALL cell line. Interestingly, while Western blot analysis revealed the hypersensitive Jkt75 substrain exhibited significantly lower baseline levels of active PAKs compared to Jkt25 cells, CC2C6 treatment did not further alter their PAK phosphorylation states. This discrepancy between proteomics and Western blot analysis may stem from the higher sensitivity of proteomic approaches compared to immunoblotting.\u003c/p\u003e \u003cp\u003eAlternatively, the INKA algorithm infers kinase activity based on the phosphorylation state of downstream proteins [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is therefore plausible that proteins involved in cytoskeletal dynamics other than PAKs were responsible for the change in protein phosphorylation assigned to PAKs by the algorithm. Indeed, INKA analysis indicated that the PAK regulator protein kinase D (PKD)\u0026mdash;which also regulates actin dynamics via modulation of slingshot and cofilin independently of PAKs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] \u0026mdash;and ROCK, a kinase whose role in actin regulation is well established [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], were also downregulated in CC2C6-treated samples. Supporting this, inhibition of these kinases (PAKs, PKD and ROCK), or Rho GTPases (which act as PAK activators but also regulate actin independently), synergized with CC2C6 in our experiments to induce increased cell death.\u003c/p\u003e \u003cp\u003e \u003cem\u003eActin dynamics and reorganization.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe actin cytoskeleton is a dynamic regulator of cell shape and is the driving force of cell motility. Consistent with previous findings [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], we confirm that cell death induced by CC2C6 is partially inhibited, and in some cases, completely inhibited, by actin depolymerization using cytochalasin D. Notably, CytoD also abolished the synergy between CC2C6 and PAK inhibition, suggesting that an intact actin structure is required for CD47-mediated death. However, we also observed that the actin stabilizer jasplakinolide inhibited CD47-mediated cell death. That both polymerization and depolymerization disrupt the process suggests that active reorganization of the actin network is a required step for the efficient induction of cell death by CD47.\u003c/p\u003e \u003cp\u003eCofilin plays an essential role in actin dynamics by severing F-actin to promote its depolymerization and turnover [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recently, Cs\u0026aacute;nyi et al. reported that ligation of CD47 by thrombospondin increased cofilin activation during micropinocytosis in macrophages [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results similarly show that cofilin activity increases upon CC2C6 treatment and is associated with higher sensitivity; the hypersensitive Jkt75 cells had significantly more activated cofilin compared to Jkt25. These data suggest a role for cofilin downstream of PAKs in regulating CD47-mediated cell death. Furthermore, because the ARP2/3 complex nucleates branched actin following cofilin-mediated severing, and WAS patients\u0026mdash;who have impaired expression of the ARP2/3 regulator WASP\u0026mdash;are insensitive to CD47-mediated cell death [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], it is likely that this death pathway requires branched actin formation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCD47 clustering and mobility.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn mature erythrocytes, CD47 is stabilized on the membrane surface by its inclusion in the band 3 macrocomplex, which links to actin via protein 4.2. However, immature erythrocytes, which lack protein 4.2, rely on the actin cytoskeleton for stability; in those cells, CytoD was found to decrease cell surface CD47 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast, CD47 remained well expressed in our T-leukemic cell models in the presence of CytoD. Therefore, the decrease in CC2C6 activity following CytoD treatment cannot be explained by the loss of surface CD47.\u003c/p\u003e \u003cp\u003eInstead, CD47 has been shown to cluster during cell death and antibody cross-linking, the latter of which induces cytoskeletal rearrangements [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, CytoD may disrupt the clustering of CD47 required for downstream cell death signaling. This parallels Fas/CD95, which also clusters during UV- or CD95-mediated apoptosis, though actin disruption did not inhibit death in some models [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, consistent with our findings, Parlato et al. found that CytoD inhibited the polarization and uropod formation associated with CD95-mediated death in T cells and in the T-ALL CEM cell line [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile CD95-mediated death is the prototypic example of apoptosis, both Fas and CD47 can induce caspase-independent phenotypes requiring RIPK [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Additionally, ezrin (an ERM family member) links CD95 to the cytoskeleton (Lozupone, Lugini et al. 2004, Hebert, Potin et al. 2008, Wajant 2014), while radixin was recently shown to directly associate with CD47 in several cancer types [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. It will be of interest to determine if CD47-mediated death depends on ERM family members or RIPK as a mediator.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell aggregation and mobility.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eDufour et al. recently found that CD47 mobility increased during apoptosis and following integrin activation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This mobility was highly dependent on cholesterol-containing lipid rafts; disrupting these rafts prevented CD47 clustering, actin rearrangements, cell spreading and SIRPα binding [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In light of our results, it will be interesting to determine if and how CC2C6-mediated cell death affects CD47 mobility and what effects CytoD might have on this.\u003c/p\u003e \u003cp\u003eWe previously reported that CD47 ligation induces cell aggregation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Here, we show that preventing this aggregation inhibits cell death, and furthermore, CD47-mediated aggregation can be inhibited by CytoD, suggesting aggregation occurs downstream of actin remodeling. Ligation of CD47 has also been linked to the phosphorylation of vascular endothelial cadherin (VEC) and increased permeability, and fibroblast aggregation has been shown to depend on CD47 homophilic interactions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given that CD47 is known to colocalize with E-cadherin, ZO-1, and cortical actin in epithelial cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], it will be interesting to determine if CD47-mediated death requires specific cell-cell adhesion structures.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis paper describes PAKs as new regulators of CD47-mediated cell death. Our data show that PAK signaling can be modulated to synergize with CD47 ligation by way of the actin cytoskeleton. Furthermore, we demonstrate that actin-dependent cell aggregation is a required step for the efficient induction of CD47-mediated cell death.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePatient-derived leukemia xenografts\u003c/h2\u003e \u003cp\u003e Studies involving human participants were conducted in accordance with the Declaration of Helsinki. Collection and use of patient-derived leukemia cells were approved by the University of British Columbia (UBC) and Children\u0026rsquo;s \u0026amp; Women\u0026rsquo;s Health Centre of British Columbia Research Ethics Board (REB) under protocol H14-02930 and H17-01860. Written informed consent was obtained from all participants or their legal guardians prior to sample collection and deposition into the BC Children\u0026rsquo;s Hospital (BCCH) Biobank. All samples were de-identified to ensure patient confidentiality before use in research.\u003c/p\u003e \u003cp\u003e All animal experiments were performed in accordance with the ethical guidelines of the Canadian Council on Animal Care (CCAC). Experimental protocols were reviewed and approved by the University of British Columbia (UBC) Animal Care Committee under protocol A19-0197.\u003c/p\u003e \u003cp\u003eCells from patient-derived xenografts were obtained by injecting NOD scid gamma (NSG) mice (Jackson Laboratory) with whole bone marrow mononuclear cells from patients and viably cryopreserving splenic cells from moribund mice. Cells used were from patients designated BT09, BT012, BT007 (T-ALLs) and BB007, BB013, BB026, BB031, BB039, BB050 (B-ALLs). For experiments, cells were thawed out and allowed to recover overnight or up to 4 days in StemSpan SFEMII with CC100 for B-ALL cells, or in StemSpan SFEMII with Immunocult Human CD3/CD28/CD2 T cell Activator and 100ng/mL IL-2 for T-ALL cells (StemCell Technologies).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCells and tissue culture\u003c/h3\u003e\n\u003cp\u003eThe following leukemic cell lines were used in this study: Jurkat T-ALL clone E6-1 (ATCC TIB-152); MOLT-4 T-ALL (ATCC CRL-1582), NALM6 B-ALL clone G5 (ATCC CRL-3273), and BV-173 chronic myeloid leukemia (DSMZ ACC 20). Jkt75 is a non-clonal natural derivative of Jurkat E6-1 (ATCC TIB-152, here also referred to as Jkt25), that through routine monitoring was found to exhibit increased CC2C6-induced cell death. Short tandem repeat (STR) analysis confirmed both Jkt75 and Jkt25 with STR profile matching ATCC TIB-152. All cells were maintained in complete RPMI (cRPMI, which is RPMI 1640 (Sigma-Aldrich) with 10% fetal bovine serum (Invitrogen), penicillin/streptomycin (Gibco), and non-essential amino acids (Invitrogen)) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and small molecule inhibitors\u003c/h2\u003e \u003cp\u003eCD47 antibodies used were clones CC2C6 (Biolegend) and B6H12 (BD Biosciences). Western blotting antibodies used were as follows: PAK1 (#2602), phospho-PAK1 (Thr423)/PAK2 (Thr402) (#2601), cofilin (#5175), and phospho-cofilin (#3313) were from CST; PAK4 (sc-393367) and phospho-PAK4 (Ser474)(sc-135775) were from Santa Cruz Biotechology; and GAPDH was from Biolegend. Secondary antibodies were goat anti-mouse DyLight488, AlexaFluor 633 or AlexaFluor plus 800, goat anti-rabbit Dylight680 or AlexaFluor plus 800, and goat anti-rat Dylight680.\u003c/p\u003e \u003cp\u003eThe pan-PAK (PF-3758309), PAK-1 (NVS-PANK-1), PAK-4 (KPT9274), CDC42 (ML-141), Rac1 (EHop-016), and PKD (CID755673) inhibitors were from Selleckchem. Additional inhibitors included Y-27632 (StemCell Technologies), cytochalasin D (Tocris or Selleckchem), and jasplakinolide (Santa Cruz Biotechnology). All were dissolved in DMSO (Sigma-Aldrich) as per manufacturer recommendations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell death assays and F-actin labelling\u003c/h3\u003e\n\u003cp\u003eCells were harvested by centrifugation, resuspended in cRPMI at 6x10\u003csup\u003e5\u003c/sup\u003e cells/mL, and incubated in a 12-well plate (Thermo) at 37\u0026deg;C with or without 250ng/mL or 500ng/mL CC2C6 for 2 hours or 24 hours. As indicated, cells may also be co-incubated with inhibitors or with DMSO vehicle control. Cells were then transferred to FACS tubes and washed in PBS. For cell death assays, samples were resuspended in binding buffer with FITC- or Cy5- conjugated annexin V according to manufacturer instructions (BD Biosciences). In some experiments, cells were incubated in a 1.5mL microcentrifuge tube with or without constant rotation using a MACSmix rotator (Miltenyi Biotec). In others, cells were plated on poly-L-lysine (Sigma)-coated 12-well plates and the cells allowed to settle for 45 minutes at 22\u003csup\u003eo\u003c/sup\u003eC before addition of CC2C6 and incubation at 37\u0026deg;C. For F-actin determination, cells were fixed and permeabilized using the BD Cytofix/ Cytoperm plus Fixation and Permeabilization kit (BD Biosciences) as per manufacturer recommendation and stained with phalloidin-FITC (Sigma-Aldrich) or pCofillin. Flow cytometry was performed using an AccuriC6 or C6plus (BD Biosciences) and data analyzed using FlowJo (BD).\u003c/p\u003e\n\u003ch3\u003eProteomics\u003c/h3\u003e\n\u003cp\u003eSamples were incubated at 37\u0026deg;C for 30 minutes with or without CC2C6, washed in PBS, split equally and centrifuged once more. Supernatants were removed and samples were snap-frozen in liquid nitrogen and stored at -80\u0026deg;C freezer until extraction for proteomics.\u003c/p\u003e \u003cp\u003eCells were lysed in buffer containing 1% SDS (Fisher BioReagents), and processed with single-pot solid-phase-enhanced sample processing (SP3). Peptide digests were washed on a C18 spin column (Nest Group Inc) with 2% acetonitrile (ACN) in 0.1% formic acid, eluted with 80% acetonitrile in 0.1% formic acid (FA) and dried in a Speed Vac. Dried samples were resuspended in 0.1% TFA in 80% ACN and phosphorylated peptides purified by immobilized metal affinity chromatography (IMAC) using Fe-NTA MagBeads (Cube Biotech). Sample solution was added to beads washed with 0.1% TFA in 80% ACN, and incubated in a thermomixer at 22\u0026deg;C for 30 min. Beads were magnetically isolated to recover the non-phosphorylated peptide fraction in the supernatant. Following two wash steps in sample resuspension buffer, phosphorylated peptides were eluted using 1% ammonia, and immediately acidified to pH 3 with FA. Peptide solutions were cleaned with C18 spin columns (Nest Group Inc) by washing with 0.1% TFA, and eluted with 0.1% FA in 50% ACN. After drying in a Speed Vac, samples were resolubilized in 0.1% FA for mass spectrometry analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLiquid chromatography tandem mass spectrometry (LC-MSMS)\u003c/h2\u003e \u003cp\u003eData-dependent Acquisition (DDA)\u0026mdash;1\u0026micro;g of phosphor-peptides were analyzed on a Q Exactive HF plus Orbitrap mass spectrometer coupled to an Easy-nLC 1200 liquid chromatography (Thermo Scientific) with a 3 cm-long homemade precolumn (Polymicro Technologies capillary tubings, 360OD, 100ID), a 35 cm-long homemade analytical column (Self-pack PicoFrit columns, 360OD, 75ID, 15 m tip ID) and packed with Dr. Maisch beads (ReproSil-Pur 120 C18-AQ, 3 um) with a flow rate at 300 nL/min and constant temperature at 50\u0026deg;C. Mobile phase A (0.1% FA in water) and mobile phase B (0.1% FA in 95% ACN) were used for a 120 min gradient. DDA: A full-scan MS spectrum (350 \u0026minus;\u0026thinsp;1600 m/z) was collected with resolution of 120,000 at m/z 200 and the maximum acquisition time of 246 ms and an AGC target value of 1e6. MS/MS scan was acquired at a resolution of 60,000 with maximum acquisition time of 118 ms and an AGC target value of 2e5 with an isolation window of 1.4 m/z at Orbitrap cell. The top 12 precursors were selected. Normalized collision energy (NCE) was set to 28. Dynamic exclusion duration was set to 15 s. Charge state exclusion was set to ignore unassigned, 1, and 5 and greater charges. The heated capillary temperature was set to 275\u0026deg;C.\u003c/p\u003e \u003cp\u003eData Processing\u0026mdash;Raw MS DDA data acquired on the Q Exactive HF were processed and searched with MaxQuant version 1.6.2.10 using the built-in Andromeda search engine. The first search peptide tolerance of 20 ppm and main search peptide tolerance of 4.5 ppm were used. The human protein database was downloaded from UniProt (release 2018_09; 20,410 sequences) and common contaminants were embedded from MaxQuant. Phosphopeptide and statistical analysis was performed in Perseus and INKA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE and western immunoblots\u003c/h2\u003e \u003cp\u003eCells were centrifuged, resuspended in cRPMI with or without treatment, and incubated at 37\u0026deg;C for the indicated time. Cells were then washed in PBS and lysed in RIPA buffer (50mM Tris pH8, 150mM NaCl, 1% Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, Complete protease inhibitors (Roche), and 25mM sodium fluoride). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Proteins were labelled with primary and secondary antibodies as indicated and membranes were imaged using the Sapphire FL Biomolecular Imager (Azure Biosystems) or the Odyssey (LICORbio) and analyzed using Image Studio (LICORbio).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy\u003c/h2\u003e \u003cp\u003eCells were centrifuged, resuspended in cRPMI with or without treatment in a 12-well plate (Thermo) and incubated at 37\u0026deg;C for 2 hours. Samples were then resuspended by pipetting thrice and cells allowed to settle for 5 minutes before imaging using an Olympus IX81 microscope (4x objective) equipped with a CoolSnap HQ2 camera (Photometrics) and controlled by Metamorph\u0026reg; software (Molecular Devices). Post-acquisition processing was performed on ImageJ.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our sincere gratitude to the patients and families for their invaluable contribution of tissue samples to the BC Children\u0026rsquo;s Hospital (BCCH) Biobank. This research was made possible by their generous participation and the expert assistance of the biobank staff in sample procurement. Arnawaz Bashir provided technical assistance at various phases of this project. Support from the BCCH Foundation for the Michael Cuccione Childhood Cancer Research Program and the BRAvE initiative enabled portions of the PDX work. This work was supported by the Canadian Institutes of Health Research (CIHR) Project Grant PJT-175116 awarded to C.J.L.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.L. and C.J.L. performed study concept and design, development of methodology and writing, review and revision of the paper; P.L., A.C.U., A.L., P.F.L. and C.J.L. performed acquisition, analysis and interpretation of data, and statistical analysis; N.R., C.A.M. and G.S.D. provided technical and material support. All authors approved the final paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSoto-Pantoja DR, Kaur S, Roberts DD. CD47 signaling pathways controlling cellular differentiation and responses to stress. \u003cem\u003eCrit Rev Biochem Mol Biol\u003c/em\u003e 2015, 50(3): 212\u0026ndash;230.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeclair P, Lim CJ. 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While actin dynamics have been implicated in this process, the upstream signaling regulators remain poorly defined. In this study, we identify p21-activated kinases (PAKs) as critical negative regulators of CD47-mediated cell death in acute lymphoblastic leukemia (ALL). Using phospho-proteomic and Inferred Kinase Activity (INKA) analysis, we observed that CD47 ligation with the monoclonal antibody CC2C6 leads to the downregulation of actin-regulatory kinases, including PAK1, PAK2, and PAK4, in patient-derived xenograft (PDX) models and Jurkat T-ALL cells. Pharmacological inhibition of PAKs using the pan-PAK inhibitor PF-3758309 markedly synergized with CD47-antibody CC2C6 to induce cell death across multiple B- and T-ALL cell lines. This synergy extended to inhibitors of other actin regulators, including ROCK, PKD, and the Rho-family GTPases, Cdc42 and Rac1. Mechanistically, we demonstrate that hypersensitivity to CD47 ligation in a novel Jurkat substrain (Jkt75) correlates with reduced basal PAK activity and increased levels of active (dephosphorylated) cofilin. We confirm that CD47-mediated death requires dynamic F-actin remodeling, as both the actin depolymerizer cytochalasin D and the stabilizer jasplakinolide significantly attenuated cell death. Furthermore, we reveal that CD47 ligation triggers robust cell-cell aggregation, which is actin-dependent and essential for the lethal signal; preventing physical cell-cell contact through rotation or immobilization effectively abolished the death response. Our findings establish a novel PAK-actin-aggregation axis that governs CD47-mediated programmed cell death. These results suggest that targeting PAK signaling may provide a potent strategy to enhance the efficacy of CD47-based immunotherapies in refractory leukemias.\u003c/p\u003e","manuscriptTitle":"Regulation of CD47-mediated cell death by p21-activated kinases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 14:51:29","doi":"10.21203/rs.3.rs-8940785/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-04-22T10:27:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-19T14:29:53+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-10T16:08:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-08T19:07:14+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-03T15:46:51+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-26T13:36:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-24T15:54:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2026-02-23T18:17:24+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-02-23T12:55:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-22T18:00:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"082e07da-374d-41a8-9881-e3843b90f7fb","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63857838,"name":"Health sciences/Diseases/Cancer/Paediatric cancer"},{"id":63857839,"name":"Biological sciences/Cell biology/Cell adhesion"}],"tags":[],"updatedAt":"2026-04-22T10:31:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 14:51:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8940785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8940785","identity":"rs-8940785","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}