Ge et al. Cell Death and Disease (2019) 10:8 https://doi.org/10.1038/s41419-018-1259-5 Cell Death & Disease

ARTICLE Open Access Midostaurin potentiates antitumor activity in Burkitt’slymphomaby inducing apoptosis Xiaowen Ge1,JianfengChen2,LingLi2,PeipeiDing2,QiWang2, Wei Zhang2,LuyingLi2,XinyueLv2, Danlei Zhou2, Zhengzeng Jiang1, Haiying Zeng1,YifanXu1,YingyongHou1 and Weiguo Hu 2

Abstract An intensive short-term chemotherapy regimen has substantially prolonged the overall survival of Burkitt’s lymphoma (BL) patients, which has been further improved by addition of rituximab. However, the inevitable development of resistance to rituximab and the toxicity of chemotherapy remain obstacles. We first prepared two BL cell lines resistant to rituximab-mediated CDC. Using a phosphorylation antibody microarray, we revealed that PI3K/AKT pathway contained the most phosphorylated proteins/hits, while apoptosis pathway that may be regulated by PKC displayed the greatest fold enrichment in the resistant cells. The PI3K/AKT inhibitor IPI-145 failed to reverse the resistance. In contrast, the pan-PKC inhibitor midostaurin exhibited potent antitumor activity in both original and resistant cells, alone or in combination with rituximab. Notably, midostaurin promoted apoptosis by reducing the phosphorylation of PKC and consequently of downstream Bad, Bcl-2 and NF-κB. Therefore, midostaurin improved rituximab activity by supplementing pro-apoptotic effects. In vivo, midostaurin alone powerfully prolonged the survival of mice bearing the resistant BL cells compared to rituximab alone treatments. Addition of midostaurin to rituximab led to dramatically fi

1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; improved survival compared to rituximab but not midostaurin monotherapy. Our ndings call for further evaluation of midostaurin alone or in combination with rituximab in treating resistant BL in particular.

Introduction intensive short-term chemotherapy regimen has sub- Burkitt’s lymphoma (BL), a highly aggressive non- stantially improved the survival rates to greater than 90% Hodgkin’s B-cell lymphoma, accounts for 3–5% of lym- in childhood BL patients4,5. Similar regimens in adult BL phoma cases in all age groups and 40–50% of all child- patients have achieved advances in outcomes, with OS – hood lymphomas1. Adult BL patients have shown a poor rates exceeding 70%6 9. Despite the success of these response to a CHOP (cyclophosphamide, doxorubicin, regimens, further progress is required to achieve a ther- vincristine and prednisolone)-based regimen, with 2-year apeutic strategy that can reduce toxicity and overcome and 5-year overall survival (OS) rates of approximately drug resistance in currently incurable patients. 50–65%, decreasing to less than 30% with bone marrow or The combination of rituximab with CHOP che- central nervous system involvement2,3. In contrast, an motherapy (R-CHOP) has improved overall survival by at least 20% in cases of diffuse large B-cell lymphoma (DLBCL)10. Similarly, many single-arm clinical trials have Correspondence: Yingyong Hou ([email protected])or confirmed the effect of adding rituximab to the intensive Weiguo Hu ([email protected]) 11–15 1Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, short-term chemotherapy regimens for BL . A recent China phase III clinical trial has shown that addition of ritux- 2 Fudan University Shanghai Cancer Center and Institutes of Biomedical imab to chemotherapy achieved better 3-year event-free Sciences; Shanghai Medical College, Fudan University, Shanghai 200032, China These authors contributed equally to this work: Xiaowen Ge, Jianfeng Chen. survival (75% vs 62%, P = 0.024) and 3-year OS (83% vs Edited by M. Diederich

© The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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70%, P = 0.011) rates than chemotherapy alone16. 640 μg/mL rituximab, respectively, and 20% NHS every Therefore, addition of rituximab is highly anticipated in 21 days to maintain resistance. The CDC effect was the future design of anti-BL regimens, and the subsequent assessed by fluorescence-activated cell sorting (FACS) development of resistance to rituximab may be pre- analysis to detect propidium iodide-positive cells. dictable as it has occurred in DLBCL17. By binding to the membrane protein CD20, rituximab Immunoblotting assay destroys lymphocytes mainly via antibody-dependent We performed immunoblotting assays according to the cellular cytotoxicity (ADCC) and complement- standard protocol using the antibodies shown in Table S1. – dependent cytotoxicity (CDC)17 19. The resistance to ADCC probably results from the intrinsic polymorphism FACS analysis of the IgG Fc receptor FcgammaRIIIa gene20, whereas the After washing with phosphate-buffered saline (PBS), resistance to CDC can most likely be attributed to the cells were incubated with fluorescein-conjugated anti- down-regulation of CD20 expression and the elevated bodies for 30 min and then rinsed and resuspended in expression of membrane complement regulatory proteins PBS. Flow cytometric analysis was performed on a (mCRPs), especially CD59 expression17,21,22. However, Cytomics FC500 MPL machine (Beckman Coulter, Brea, many studies have revealed that rituximab fails to induce CA) and analyzed with the FlowJo software (Ashland, apoptosis to any detectable extent in B-cell lymphoma, OR). We performed cell sorting with a MoFlo XDP – including in BL cells23 30. Therefore, the development of instrument (Beckman Coulter, Brea, CA) based on a pro-apoptotic agent to combine with rituximab is a fluorescence. rational approach to achieving either high anti-cancer Apoptosis analysis was performed using the PE Annexin efficacy with rituximab or overcoming the resistance to V Apoptosis Detection Kit (BD Pharmingen, San Diego, rituximab. To identify such an alternative therapeutic CA) according to the manufacturer’s instructions. approach, we prepared two BL cell lines resistant to rituximab-mediated CDC, interrogated the signaling CytoTox-Glo™ cytotoxicity assay pathways related to the development of resistance, and We used the CytoTox-Glo™ cytotoxicity assay kit evaluated the effect of pathway inhibitors on antitumor (Promega, Madison, WI) to detect cytotoxicity. The cells activity and overcoming resistance. were pretreated with rituximab and 20% NHS in the presence or absence of midostaurin for 48 h before assays. Materials and methods Cytotoxicity was calculated according to the formula: Cell culture and reagents cytotoxicity (%) = dead cell luminescence/total lumines- Two BL cell lines, Raji and Ramos, were purchased from cence × 100%. American Type Culture Collection (Manassas, VA) and were maintained in RPMI-1640 medium supplemented Antibody microarray for profiling phosphorylation with 10% fetal bovine serum (GIBCO BRL, Grand Island, Protein phosphorylation was profiled using a NY) and 1% penicillin/streptomycin (Ambion, Austin, phosphorylation-profiling antibody microarray (Full TX). Moon Microsystems, Catalog No. CSP100, Sunnyvale, As a complement resource, normal human serum CA) containing 269 antibodies against 131 protein phos- (NHS) was pooled from 10 healthy persons, aliquoted and phorylation sites; the procedure was performed by Wayen stored at −80 °C until use. The phosphoinositide 3-kinase Biotechnology (Shanghai, China) according to the estab- (PI3K) inhibitor IPI-14531 and the protein kinase C (PKC) lished protocol. Differentially phosphorylated proteins inhibitor midostaurin were purchased from Selleck Che- with a fold change >1.5 were screened by DAVID (Data- micals (Houston, TX), and dissolved in dimethyl sulfoxide base for Annotation, Visualization and Integrated Dis- (DMSO) in use. Consequently, same volume DMSO was covery) to performed functional annotation33. The used as control. interaction network among PKC subunits and the differ- entially phosphorylated proteins was analyzed using Generation of rituximab-resistant BL cells Ingenuity Pathway Analysis (IPA, QIAGEN, Dusseldorf, We developed Raji and Ramos cells that were resistant Germany). to rituximab-mediated CDC as previously described32. Briefly, the original Raji or Ramos cells were treated with RNA sequencing escalating rituximab (Roche, Basel, Switzerland) con- RNA sequencing (RNA-seq) was performed as pre- centrations from 4 or 40 μg/mL to 32 or 640 μg/mL, viously described34. Briefly, total RNA was extracted and respectively, in the presence of 20% NHS. The resulting pooled separately from three different passages of Ramos resistant cells were termed Raji32 and Ramos640, and Ramos640 cells. The RNA quality was determined respectively. These cells were treated with 32 μg/mL or with a Bioanalyzer 2200 (Agilent Technologies, Santa,

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Clara, CA). RNA with an RNA integrity number (RIN) Laboratory Animal Co. (Shanghai, China). Raji32-Luc >8.0 was considered acceptable for complementary DNA cells were resuspended in PBS and then injected intra- (cDNA) library construction. The RNA-seq was con- peritoneally at 1.5 × 107 cells per mouse. The mice were ducted by Shanghai Novelbio, Ltd. cDNA libraries were divided into 4 groups (7 mice per group) based on the constructed using the Ion Total RNA-seq Kit v2.0 (Life administered drugs, i.e., saline, rituximab, midostaurin Technologies, Gaithersburg, MD) and were then pro- and rituximab plus midostaurin. Rituximab was intra- cessed for RNA sequencing. Before the read mapping, peritoneally injected at 118.4 mg/kg on days 8, 12 and 16, clean reads were obtained from the raw reads by removing and midostaurin was administered by gavage at 20 mg/kg adaptor sequences, and these reads were then aligned to on days 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 the human genome (version: GRCh37 NCBI) using the after implantation. Saline was administered in the same MapSplice program (v2.1.6). After the significance and way as rituximab with the same injection volume. Tumor false discovery rate (FDR) analyses were performed under growth was monitored by bioluminescence at 50, 70 and the following criteria: (1) fold change >1.5 or <0.667 and 90 days after implantation. For the in vivo luminescence (2) FDR <0.05, we applied the DEseq algorithm to filter imaging, D-luciferin (Promega, Madison, WI) was intra- the differentially expressed genes. peritoneally injected (150 mg/kg). After 10 min, the mice were anesthetized by intraperitoneal injection with pen- Gene set enrichment analysis tobarbital (50 mg/kg), and bioluminescence was then Using gene set enrichment analysis (GSEA) software visualized using an In-Vivo MS FX PRO system (Bruker, (the Broad Institute at MIT), we performed a GSEA to Billerica, MA). The survival time of each mouse was identify the functions of the differentially expressed genes recorded until 120 days. All the animal experiments were obtained from the RNA-seq35,36. The pre-ranked version conducted in strict accordance with experimental proto- of the software was used to identify significantly enriched cols approved by the Animal Ethics Committee at pathways, and enriched pathways with an FDR <0.25 were Shanghai Medical School, Fudan University. considered significant. The anti-apoptotic TNFs/NF-κB/ Bcl-2 pathway gene set used in this study consisted of 42 Statistics genes from the ‘apoptosis and survival anti-apoptotic The data are presented as the mean ± SD unless TNFs/NF-κB/Bcl-2 pathway SuperPath’ in the PathCards otherwise specified. The significance of differences pathway unification database (Version 4.6.0.37, Weiz- between two groups was determined using the two-tailed mann Institute of Science). The p53 pathway gene set Student’s t-test for unpaired data. For the total photon used in this study consisted of 132 genes from the ‘p53 flux in the animal models, the significance of differences pathway (RnD) SuperPath’ in the PathCards pathway was determined using the one-tailed Mann–Whitney test. unification database. We applied the Mantel–Cox test to compare the survival rates of two groups of xenograft mouse models. In all Plasmid construction and lentiviral transduction analyses, P < 0.05 was considered statistically significant. The Coding Sequence (CDS) of the firefly luciferase gene was obtained by PCR amplification from the pGL3- Results Basic plasmid and inserted into the pCDH cDNA cloning Reduced CD20 and elevated CD59 expression levels led to and expression lentivector. Primers for the firefly lucifer- the resistance of BL cells to rituximab-mediated CDC ase CDS amplification were as follows: forward primer 5′- The intrinsic features of immune cells precipitate the ATGGAAGACGCCAAAAACATAAAG-3′, reverse pri- resistance to rituximab-mediated ADCC20, while apop- mer 5′-TTACACGGCGATCTTTCCGCCCTT-3′. The tosis plays a negligible role in rituximab antitumor pCDH plasmid was co-transfected with the pMD.2G and activity. Therefore, we generated two BL cell lines, psPAX2 plasmids into 293FT cells to generate a firefly Ramos640 and Raji32, that were resistant to rituximab- luciferase overexpression lentivirus. The lentivirus was mediated CDC at rituximab concentrations of 640 and 32 subsequently added to the culture medium of Raji32 cells μg/mL, respectively. Using immunoblotting (Fig. 1a, c) for 48 h of incubation. All the cells transfected with the and FACS (Fig. 1b, d) assays, we found that CD20 lentivirus in this study were sorted based on green expression decreased while CD59 expression increased in fluorescent protein using a MoFlo XDP instrument both resistant cell lines compared to the expression levels (Beckman Coulter, Brea, CA) and were termed Raji32-Luc in the corresponding original cells (Fig. 1). However, the cells. expression levels of two other mCRPs were not consistent between the two resistant cell lines. CD55 expression was Xenograft model reduced in Ramos640 cells but was increased in Raji32 The 8-week-old female SCID (severe combined immu- cells, and CD46 expression was unchanged in Ramos640 nodeficiency) mice were purchased from SLAC cells but was reduced in Raji32 cells (Fig. 1). These results

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Fig. 1 The expression levels of CD20 and mCRPs in resistant BL cells. a–d Compared to the original cells, CD20 expression was reduced, while CD59 expression was elevated in the resistant Ramos640 (a, b) and Raji32 (c, d) cells as determined by western blot analysis (a, c) and FACS (b, d). However, CD55 expression was slightly reduced in the resistant Ramos640 cells (a, b), but significantly increased in the resistant Raji32 cells (c, d). In addition, CD46 expression was only decreased in the resistant Raji32 cells (c, d) but not in the Ramos640 cells (a, b). The data are presented as the means ± SD, n = 3, NS no significance, **P < 0.01, and ****P < 0.0001

Table 1 Enriched pathways in Ramos640 vs Ramos cells based on an antibody microarray for profiling phosphorylation

KEGG pathway P value Fold enrichment Count Genes hsa04151: PI3K-Akt signaling pathway 2.47E−15 18.60 13 CDKN1A, PTK2, CDKN1B, BCL2, CREB1, TP53, BAD, BCL2L1, ITGB3, PTEN, MYC, CHUK, AKT2 hsa05200: Pathways in cancer 1.24E−12 15.07 12 CDKN1A, PTK2, CDKN1B, JUN, BCL2, TP53, BAD, BCL2L1, PTEN, MYC, CHUK, AKT2 hsa04012: ErbB signaling pathway 5.35E−09 39.71 7 CDKN1A, PTK2, CDKN1B, JUN, BAD, MYC, AKT2 hsa05169: Epstein-Barr virus infection 1.59E−08 20.78 8 CDKN1A, CDKN1B, JUN, BCL2, TP53, MYC, CHUK, AKT2 hsa04210: Apoptosis 6.01E−08 47.76 6 BCL2, TP53, BAD, BCL2L1, CHUK, AKT2 hsa04510: Focal adhesion 9.41E−07 16.77 7 PTK2, JUN, BCL2, BAD, ITGB3, PTEN, AKT2 hsa05206: microRNAs in cancer 6.28E−06 12.12 7 CDKN1A, CDKN1B, BCL2, TP53, ITGB3, PTEN, MYC hsa05202: Transcriptional misregulation in 8.80E−06 17.63 6 CDKN1A, PTK2, CDKN1B, TP53, BCL2L1, MYC cancer hsa05205: Proteoglycans in cancer 2.06E−05 14.81 6 CDKN1A, PTK2, TP53, ITGB3, MYC, AKT2 hsa05203: Viral carcinogenesis 2.32E−05 14.45 6 CDKN1A, CDKN1B, JUN, CREB1, TP53, BAD

are consistent with previous reports showing that reduced clinical trial (phase I) to determine their safety in B-cell CD20 and elevated CD59 levels contribute to the devel- non-Hodgkin lymphoma (NHL) patients41, and no further opment of resistance to rituximab-mediated CDC17,21,22. clinical trials have been reported. Therefore, alternative Considerable efforts have been made to improve ritux- therapeutic strategies are still required. imab therapeutic efficacy by increasing CD20 expression, for example, by administering the histone deacetylase PI3K/Akt pathway was strongly enriched in the resistant inhibitor trichostatin A37 or synthetic CpG oligodeox- cells, but its inhibition failed to reverse the resistance ynucleotides38 or by inhibiting CD59 function with a Considering the negligible effect of rituximab on indu- modified monoclonal antibody39,40 or the bacterial toxin- cing apoptosis in B-NHL, addition of a pro-apoptotic derived ILYd432. However, among these approaches, only agent to rituximab therapy would be an ideal strategy. CpG oligodeoxynucleotides were further evaluated in a Therefore, an antibody microarray for phosphorylation

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Fig. 2 PI3K/Akt inhibition failed to enhance the susceptibility of BL cells to rituximab-mediated CDC. a The phosphorylation levels of Akt (S473) increased in the resistant Ramos640 and Raji cells compared to the corresponding original cells, which could be suppressed by treatment with the PI3K inhibitor IPI-145, leading to reduced expression of CD20 and CD55. b The effect of combining IPI-145 with rituximab on rituximab-mediated CDC. IPI-145 alone failed to increase cell death in all the original and resistant cells except for the Raji32 cells. The addition of IPI-145 to rituximab also failed to increase rituximab-mediated CDC in the Ramos and Raji cells, but not in the Raji32 cells. In contrast, this combination treatment significantly increased rituximab-mediated CDC in the Raji32 cells. The data are represented as the means ± SD, n = 3, NS no significance, ***P < 0.001, ****P < 0.0001. RTX rituximab, NHS normal human serum profiling was used to identify the functional proteins and (Fig. 2a). These outcomes subsequently resulted in no signaling pathways activated in Ramos640 cells. Fluores- effect or even a slightly negative effect of IPI-145 on the cence analysis revealed that the phosphorylation levels of enhancement of rituximab-mediated CDC in the original 35 proteins were up- (31/35) or down-regulated (4/35) by and resistant cells, with the exception of Raji32 cells greater than 1.4-fold in Ramos640 cells compared to the (Fig. 2b). In the Raji32 cells, IPI-145 alone or in combi- levels in Ramos cells (Supplementary Data 1). KEGG nation with rituximab slightly enhanced cell death com- (Kyoto Encyclopedia of Genes and Genomes) signaling pared to that observed in the control or rituximab alone pathway analysis of these proteins demonstrated that treatments, respectively (Fig. 2b, right panel). These PI3K/AKT signaling pathway contained the most phos- results suggest that PI3K/Akt might not be a valuable phorylated proteins/hits, with 13, while apoptosis pathway drug target for facilitating rituximab treatment. displayed the greatest fold enrichment, at 47.76 (Table 1). Next, the PI3K inhibitor IPI-145 was used to determine The PKC-mediated apoptosis pathway was highly whether PI3K inhibition could increase the susceptibility activated in resistant cells of resistant BL cells to rituximab treatment. We first Although apoptosis pathway was identified as the most confirmed that PI3K/Akt was activated in Ramos640 and enriched in the antibody microarray, only six proteins Raji32 cells compared to the corresponding original cells were included in this pathway (Table 1), which hindered and that IPI-145 effectively impaired the phosphorylation the isolation of the pathway modulators. Therefore, we of Akt (Fig. 2a). However, CD20 expression was strongly narrowed the scope of the investigation by elevating the suppressed by IPI-145 treatment in both original and fold change of up- and down-regulated phosphorylated resistant cells, whereas the expression levels of three proteins/sites to 1.5 in the phosphorylation-profiling mCRPs CD59, CD55 and CD46 were nearly unchanged antibody microarray. The results revealed that a total of

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Fig. 3 Bioinformatics analysis revealed the anti-apoptotic effect regulated by PKC signaling in Ramos640 cells. a Phosphorylated proteins/ hits up-regulated by greater than 1.5-fold in an antibody microarray for profiling phosphorylation. b IPA: the distinct PKC isoforms regulate apoptosis via the up-regulated proteins/hits in (a). c, d GSEA for TNFs/NF-κB/Bcl-2 (c) or p53 (d) signatures in comparing the RNA-Seq data of the Ramos and Ramos640 cells. Results with an FDR < 0.25 were considered to be significant. Solid lines represent direct regulation, and dotted lines represent indirect regulation

16 proteins/sites were up-regulated by greater than 1.5- pathway was strongly elevated, while the expression of fold (Fig. 3a, and Supplementary Data 1). Notably, all of genes in p53 pathway was significantly reduced in these up-regulated proteins/sites were determined to be Ramos640 cells compared to the levels in Ramos cells involved in apoptosis according to the IPA (Fig. 3b). More based on the GSEA (Fig. 3c, d, and Supplementary importantly, the IPA also revealed that PKC signaling Data 2). This analysis also indicated that the anti- regulates all of these up-regulated proteins (Fig. 3b), apoptotic genes were up-regulated, while the pro- although the detection of PKC phosphorylation was not apoptotic genes were down-regulated. Therefore, these included in the antibody microarray set used to profile data suggested that PKC phosphorylated multiple down- phosphorylation. Using RNA-seq assay, we further found stream proteins, leading to the anti-apoptotic effect in that the expression of genes in TNFs/NF-κB/Bcl-2 Ramos640 cells.

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Fig. 4 PKC inhibition strongly induced apoptosis in both the original and resistant BL cells. a The indicated PKC isoforms were up-regulated in the resistant BL cells, leading to elevated levels of PKC phosphorylation and the activation of downstream anti-apoptotic proteins. b, c The pan-PKC inhibitor midostaurin potently induced apoptosis in the original and resistant Ramos (b) and Raji (c) cells, and the addition of rituximab to midostaurin significantly enhanced the pro-apoptotic effect in the Ramos640 cells. The data are presented as the means ± SD, n = 3, **P < 0.01, ***P < 0.001, ****P < 0.0001. RTX rituximab (640 or 32 μg/mL for Ramos or Raji, respectively), Mido midostaurin (1 μM)

The pan-PKC inhibitor midostaurin strongly promoted (SM)42,43. We observed that midostaurin suppressed the apoptosis phosphorylation levels of PKC in both resistant BL cell We compared the expression levels of five PKC iso- lines and original Ramos cells but not original Raji cells forms in the original and resistant BL cells, and found that and that this kinase inhibitor reduced the expression PKCα/β2/γ/η in Ramos640 and PKCα/β1/β2/η in Raji32 levels of PKCβ2/η in Ramos cells, of PKCα/η in Ramos640 cells were overexpressed compared to the levels in the cells and of PKCβ1/η in Raji32 cells (Fig. 4a). corresponding original cells. More importantly, the Next, we verified the phosphorylation levels of several phosphorylation levels of PKC, which were detected by PKC downstream signaling molecules, including Bad, Bcl- the sole commercially available antibody recognizing all 2 and NF-κB subunit p65. We first found that the phos- PKC isoforms, were increased in both resistant cell lines phorylated levels of these molecules were significantly (Fig. 4a). Further, we detected the efficacy of midostaurin elevated (Fig. 4a). Furthermore, the expression levels of in regulating PKC phosphorylation. Midostaurin is a Bad were decreased, while those of p65 were increased in multi-kinase inhibitor that was initially targeted toward both resistant cell lines (Fig. 4a). All of the above changes PKC and is currently approved for acute myelogenous may contribute to anti-apoptosis to a considerable degree, leukemia (AML) with an Fms-like tyrosine kinase 3 thus leading to the development of rituximab resistance in (FLT3) mutation and advanced systemic mastocytosis Ramos640 and Raji32 cells. In addition, midostaurin

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Fig. 5 Midostaurin additively enhanced rituximab-mediated CDC in resistant BL cells. a The PKC inhibitor significantly reduced CD20 expression in both the original and resistant cells and CD59 expression mainly in the resistant cells but did not alter the expression of CD55 or CD46 in the original or resistant cells. b, c The effect of the PKC inhibitor on rituximab-mediated CDC. The addition of the PKC inhibitor failed to enhance rituximab-mediated CDC in the original Ramos (b) and Raji (c) cells; however, the inhibitor additively enhanced CDC in the resistant Ramos640 (b) and Raji32 (c) cells. The data are presented as the means ± SD, n = 3, ****P < 0.0001. RTX rituximab (40 or 4 μg/mL for Ramos or Raji, respectively; or 640 or 32 μg/mL for Ramos640 or Raji32, respectively), NHS normal human serum (20%), Mido midostaurin (1 μM) suppressed the phosphorylation levels of Bad, Bcl-2 and indicated that, in general, Raji cells were more resistant to p65 in all the original and resistant cells (Fig. 4a). More- midostaurin-induced apoptosis than Ramos cells. In over, midostaurin increased or reduced the expression of addition, the combination of midostaurin with rituximab Bad or p65, respectively, in all four original and resistant resulted in slight pro-apoptotic effects only in the original cell lines and reduced the expression of Bcl-2 only in the but not in the resistant Ramos and Raji cell lines (Fig. 4b, original and resistant Raji cells (Fig. 4a). Therefore, mid- c), further supporting the conclusion that rituximab ostaurin inhibited PKC and its subsequent downstream induces a negligible pro-apoptotic effect, if any. signaling molecules, which may lead to the pro-apoptotic effect in both Ramos and Raji cells to varying degrees. Midostaurin additively enhanced the susceptibility of Among the cell lines, Raji cells appeared to be more resistant BL cells to rituximab-mediated CDC resistant to midostaurin-mediated PKC inhibition than Rituximab-mediated CDC can be regulated by the Ramos cells because midostaurin failed to suppress PKC expression of mCRPs, such as CD46, CD55 and especially phosphorylation. CD59, in addition to CD2017,22,44. We observed that Furthermore, we assessed the pro-apoptotic effects of midostaurin reduced the expression levels of CD20 in all midostaurin alone or in combination with rituximab on four BL cell lines and the levels of CD59 mainly in the the original and resistant BL cells. As we expected, resistant cells, while this inhibitor had no effect on the treatment with rituximab alone failed to induce apoptosis expression levels of CD55 or CD46 (Fig. 5a). These results in all four original or resistant BL cell lines (Fig. 4b, c). suggested that midostaurin may hinder rituximab- However, compared to the culture medium control, mediated CDC due to the reduced expression of only midostaurin alone dramatically induced cell apoptosis in CD20 in the original BL cells, while the effect of mid- Ramos (from 4.7 to 63.3%), Ramos640 (from 10.8 to ostaurin on the resistant BL cells requires further exam- 50.2%) and Raji32 (from 6.4 to 25.4%) cells and slightly ination due to the reduced expression of both CD20 and increased apoptosis (from 6.6% to 10.9%) in Raji cells, CD59. In addition, these results revealed a difference although the P value did reach statistical significance (P = between the IPI-145 and midostaurin effects on regulating 0.0038) (Fig. 4b, c). These results also functionally CD20 and CD59 expression, in which IPI-145 only

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Fig. 6 Combination treatment with midostaurin and rituximab significantly suppressed the tumor growth of the resistant Raji32 cells. Tumor masses are represented by the intensity of firefly luciferase activity (a, c, e), with the quantitative results of total photon flux shown in (b, d, f). g The survival curves of mice that were implanted with Raji32-Luc cells and treated with the indicated drugs. The data are presented as the means ± SEM (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001. A red 'X' represents mouse death in (c and e). RTX rituximab (intraperitoneally injected at 118.4 mg/kg on days 8, 12 and 16), Mido midostaurin (administrated by gavage at 20 mg/kg/day on days 8–21 for total 14 days)

reduced CD20 but not CD59 expression in the resistant Combination treatment of rituximab with midostaurin BL cells (Fig. 2a). significantly suppressed tumor growth of the resistant While rituximab plus NHS potently induced CDC in the cells original Ramos and Raji cells, addition of midostaurin Given that the potent pro-apoptotic effect of mid- failed to enhance the susceptibility to rituximab-mediated ostaurin may help increase rituximab antitumor activity, CDC, although midostaurin promoted apoptosis (Fig. 5b, we tested the efficacy of a combination treatment of c). This result may be caused by the reduced CD20 rituximab with midostaurin in immunodeficient mice that expression and the already high cell death rate induced by were implanted with the more resistant Raji32 cells treatment with rituximab alone. In contrast, addition of transfected with a luciferase-expressing plasmid. On day rituximab significantly enhanced the cytotoxic effect of 50 after implantation, the tumor growth represented by midostaurin in both original BL cell lines (Fig. 5b, c), total flux was ranked in the order of saline, rituximab, indicating that rituximab-mediated CDC dominated over midostaurin and rituximab plus midostaurin, in which all midostaurin-induced apoptosis in the original BL the adjacent groups exhibited statistically significant dif- cells. Notably, we found that the cytotoxic effect of the ferences in tumor mass (Fig. 6a, b). On day 70, 3 out of 7 combined rituximab and midostaurin treatment mice died in the saline group, 1 out of 7 died in the appeared to be additive in the resistant Ramos640 and rituximab group, and all 7 mice survived in the mid- Raji32 cells. Rituximab and midostaurin induced 17.1 and ostaurin and combination treatment groups (Fig. 6c). The 54.3% cell death, respectively, while the combination tumor mass in the surviving mice showed consistent induced 72.8% cell death (Fig. 5b). Similarly, rituximab results (Fig. 6d). On day 90, 1 out of 7, 4 out of 7, or 6 out and midostaurin induced 28.0 and 25.8% cell death, of 7 mice survived in the saline or rituximab groups, respectively, while their combination induced 62.9% cell midostaurin group, or combination treatment group, death (Fig. 5c). These results may be due to the distinct respectively (Fig. 6e). The tumor mass in the surviving antitumor mechanisms of rituximab and midostaurin. mouse with saline treatment was greater than that in the

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surviving mouse with rituximab treatment (Fig. 6f). In implemented to sensitize rituximab therapy by combining addition, the survival curve analysis demonstrated that with other agents to increase the pro-apoptotic effect. compared to rituximab alone, midostaurin alone or in These additional agents include macromolecules such as combination with rituximab significantly prolonged the the humanized monoclonal mapatumumab targeting survival rate (Fig. 6g), indicating that the pro-apoptotic TRAIL-R149, the genetically engineered fusion proteins effect induced by midostaurin may be a necessary and scFvRit:sFasL50, Apo2 ligand (Apo2L)/TRAIL (dula- beneficial supplement to the therapeutic regimen, espe- nermin)51,52 and anti-CD20-interleukin-2153, and small cially for the treatment of resistant BL. molecules, such as the selective NEDD8 activating enzyme inhibitor pevonedistat (MLN4924)54, the mTOR Discussion (mammalian target of rapamycin) inhibitor temsir- In the present study, we revealed that PKC was highly olimus55, and the proteasome inhibitor bortezomib56. up-regulated and activated in rituximab-resistant BL cells. However, the efficacy of these agents requires further The administration of the pan-PKC inhibitor midostaurin confirmation or has been suggested to be insufficient alone or in combination with rituximab potently elicited based on clinical trials52. Interestingly, apoptosis and dramatically improved OS, especially in (GA101), a novel type II glycol-engineered humanized resistant BL cells. Although the function of PKC in cancer monoclonal anti-CD20 IgG1 antibody, has shown its development remains controversial45, PKC inhibitors are superior efficacy against CLL (chronic lymphocytic leu- being widely tested for the treatment of multiple types of kemia) and relapsed/refractory indolent NHL to ritux- cancer in clinical trials. In April 2017, midostaurin was imab57,58, due to the enhanced capability of inducing approved for the treatment of AML with the FLT3 direct cell death and ADCC, though less potent in med- mutation and advanced SM42,43,46. Currently, no clinical iating CDC59. Moreover, the similar superior efficacy of trials for BL treatment are registered at clinicaltrials.gov, obinutuzumab to rituximab has also been observed in although there have been a small number of studies rituximab-sensitive/resistant BL60,61. Although the reporting the importance of PKC activation in BL underlying mechanisms by which obinutuzumab is able to cells47,48. Therefore, our findings suggest that it is induce strong direct cell death remain largely obscure, imperative to perform a clinical trial of midostaurin alone several mediators have been suspected including reactive or in combination with rituximab, preferably for relapsed/ oxygen species27, BCR (B cell antigen receptor) or cyto- refractory BL patients and possibly for other relapsed/ toxicity pathway61. Therefore, whether addition of mid- refractory NHL patients with highly activated PKC. ostaurin to obinutuzumab could enhance the antitumor Rituximab has achieved great success in the treatment activity is worthy of future investigation. Herein, we of a broad variety of B-cell lymphomas. Rituximab may reported that midostaurin strongly enhanced rituximab destroy CD20-expressing lymphocytes mainly via ADCC activity by promoting apoptosis via PKC inhibition in BL – and CDC by binding to the CD20 on the membrane17 19. cells. However, resistance to rituximab remains a major chal- Midostaurin was modified from staurosporine to lenge for relapsed/refractory patients. Approximately 50% improve the selectivity toward PKC and was then further of patients are unresponsive to rituximab treatment found to inhibit other kinases, such as FLT3, PDGFR, KIT despite CD20 expression, and initially responsive patients and VEGFR2, together with the midostaurin metabo- eventually develop resistance to further rituximab treat- lites62. Midostaurin was found to be safe and tolerable ment17,18. It may be not possible to overcome the resis- when administered as a chronic oral therapy in a phase I tance to ADCC because this resistance likely results from clinical trial63 and was approved by the FDA (Food and the intrinsic immune features of patients20. Therefore, the Drug Administration) based on its effectiveness64. Con- current approaches mainly focus on enhancing the effi- sidering that PKC was highly phosphorylated in the cacy of rituximab-mediated CDC, including up-regulating resistant BL cells, we tested the in vitro and in vivo effects CD20 expression using the histone deacetylase inhibitor of midostaurin on potentiating rituximab trichostatin A37 or synthetic CpG oligodeoxynucleotides38 antitumor activity in the original and resistant BL cells. and inhibiting CD59 function using a modified mono- We found that midostaurin strongly enhanced rituximab clonal antibody39,40 or a bacterial toxin-derived ILYd432. cytotoxicity, especially in the resistant BL cells, by pro- However, these effects observed in experimental systems moting apoptosis, possibly by altering the phosphoryla- require further careful investigation, including an assess- tion of downstream signaling molecules, including Bad, ment of their toxicity. Bcl-2 and NF-κB. Our findings support the further eva- Although a direct rituximab-induced cytotoxic effect on luation in clinical trials of midostaurin alone or in com- lymphocytes is somewhat controversial, numerous studies bination with rituximab and with or without short-term in various NHL cells have demonstrated that rituximab intensive chemotherapy in relapsed/refractory BL lacks pro-apoptotic capability. Some strategies have been patients.

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