Copanlisib Promotes Growth Inhibition and Apoptosis by Modulating The

Yan et al. Cell Death and Disease (2020) 11:943 https://doi.org/10.1038/s41419-020-03154-w Cell Death & Disease

ARTICLE Open Access Copanlisib promotes growth inhibition and apoptosis by modulating the AKT/FoxO3a/PUMA axis in colorectal cancer Ji Yan1, Shida Yang2,HongTian3,YangZhang4 and Hongmei Zhao2

Abstract Colorectal cancer (CRC) is the type of cancer with the third highest incidence and is associated with high mortality and low 5-year survival rates. We observed that copanlisib, an inhibitor of PI3K (pan-class I phosphoinositide 3-kinase) that preferentially inhibits PI3Kδ and PI3Kα, impedes the growth of CRC cells by inducing apoptosis via PUMA. There was a marked increase in the expression of PUMA independent of p53 after treatment with copanlisib. The response of CRC cells to copanlisib could be predicted by PUMA expression. Copanlisib was found to induce PUMA expression through FoxO3a by directly binding to the PUMA promoter after inhibiting AKT signaling. PUMA deﬁciency mitigated the apoptosis induced by copanlisib. Caspase activation and mitochondrial dysfunction led to copanlisib resistance, as observed through a clonogenic assay, whereas enhanced expression of PUMA increased the copanlisib-induced susceptibility to apoptosis. Moreover, the antitumor effects of copanlisib were suppressed by a deﬁciency of PUMA in a xenograft model, and caspase activation and reduced apoptosis were also observed in vivo. Copanlisib-mediated chemosensitization seemed to involve the concurrent induction of PUMA expression via mechanisms that were both dependent and independent of p53. These observations indicate that apoptosis mediated by PUMA is crucial for the anticancer effects of copanlisib and that manipulation of PUMA may aid in enhancing anticancer activities. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,;

Introduction niﬁcant improvement in the 5-year survival rate of Clinically, colorectal cancer (CRC) is a frequently CRC7,8. Treatment of CRC faces the major challenge of occurring and widely observed malignancy1,2. Worldwide, tumor neovascularization, which often causes CRC CRC is the third and second most common cancer in metastasis and invasion of other organs9. male and female patients, respectively, and its mortality Copanlisib (also known as BAY 80-6946) is a pan-class I rate is the fourth and third highest in these groups, PI3K IV inhibitor with dominant activity toward PI3Kα respectively3,4. In the past 20 years, there have been and PI3Kδ10. Copanlisib has also exhibited preclinical annual increases in the incidence and mortality rates of antitumor activity in diffuse large B cell lymphoma CRC5,6. Although postoperative prevention and therapy (DLBCL) and chronic lymphocytic leukemia (CLL)11. The has led to better CRC treatments, there has been no sig- early demonstration of the clinical activity of copanlisib in FL and DLBCL was conﬁrmed in phase 2 studies and extended to MZL, MCL, PTCL (peripheral T cell lymphoma), and small lymphocytic lymphoma12. The toxicity associated with copanlisib compares well with that asso- Correspondence: Hongmei Zhao ([email protected]) ciated with other agents of the same class, and copanlisib 1Department of Medicine Laboratory, The 4th People’s Hospital of Shenyang, Shenyang, Liaoning, China is associated with fewer and less severe gastrointestinal 2Department of Laboratory Medicine, The People’s Hospital of China Medical toxicities than idelalisib10,13. Copanlisib is now approved ’ University (The People s Hospital of Liaoning Province), Shenyang, Liaoning, by the FDA for relapsed FL patients after at least China Full list of author information is available at the end of the article Edited by I. Amelio

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Table 1 Genes mutations in CRC cell lines. Materials and methods Cell culture and treatment Cell line PIK3CA PIK3R1 p53 Human colorectal cancer cell lines (DLD1, HCT116, HCT116 Mut WT WT HT29, RKO, Lim2405, SW620, SW837, Lim1215, HCT-8, SW48, SW480, SW1463, HCT15, and LoVo) were DLD1 Mut WT WT obtained from ATCC (Manassas, VA). The status of SW1463 WT WT Mut PIK3CA, PIK3R1, and p53 in the cell lines mentioned Lim1215 WT WT WT above is listed in Table 1. The CRISPR/Cas9 system was p53 PUMA HCT15 Mut WT WT used to generate -KO (knockout) and -KO HCT116 cells. Each cell line was cultured in modified HT29 Mut WT Mut McCoy’s 5A media from Invitrogen (Carlsbad, CA) con- RKO Mut WT WT taining 10% FBS from HyClone (Logan, UT), one hundred Lim2405 WT WT WT units per milliliter penicillin, and one hundred micro- SW620 WT WT Mut grams per milliliter streptomycin from Invitrogen. NCM356 and NCM460 cells were procured from INCELL SW48 Mut WT WT and grown in the provided enriched M3 base medium SW480 WT WT Mut (M310A; INCELL) supplemented with 5% FBS and anti- LoVo WT WT WT biotics in a 37 °C incubator with 5% CO2. All of the cell SW837 WT WT Mut lines listed above were authenticated by short tandem repeat profiling and were routinely tested for mycoplasma HCT-8 Mut WT WT contamination. Twenty-four hours before drug treatment, cells (20–30% density) were added to 12-well plates. The reagent stocks of copanlisib, 5-FU, and regorafenib in 2 systemic therapies due to the results of a phase 2 study dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO) were showing an ORR of 59% and a CR of 14%14. diluted to the required concentrations in the media used PUMA (p53-upregulated modulator of apoptosis) is a for cell culture. member of the family of BH3-only Bcl-2 proteins and a strong inducer of apoptosis15. In response to the DNA Knockout cell line generation with CRISPR/Cas9 damage caused by gamma radiation and common che- The website for CRISPR design (http://crispr.mit.edu/), motherapy drugs, p53 activates the transcription of provided by the Feng Zhang Lab, was used to design the PUMA16. PUMA causes permeabilization of the mito- CRISPR/Cas9 guide RNA (gRNA) sequences. The oligo- chondrial membrane, subsequent activation of the caspase nucleotides complementary for the FoxO3a or PUMA cascade by binding to each of the five members of the gRNA were cloned after annealing into the pX459 antiapoptotic Bcl-2 family, including Bcl-2 and Bcl-XL, CRISPR/Cas9-Puro vector from Addgene (Cambridge, and release of Bax and Bak inhibition17. PUMA knockout MA). The transfection of DLD1 and HCT116 cells was causes resistance to the genotoxic agent-induced, p53- performed with either pX459/gRNA-FoxO3a or pX459/ dependent apoptosis in human cancer cells and mice18,19. gRNA-PUMA using Lipofectamine 2000, as per the pro- However, abnormalities of p53 cause dysfunctional p53- vided instructions. Twenty-four hours posttransfection, dependent regulation of PUMA in most cancer cells, the cells were treated with puromycin (2 μg/mL) for 72 h. leading to tumor cell survival and resistance to treat- After 14 days, the isolation of colonies using cloning ment20. PUMA-mediated apoptosis that is independent of cylinders was performed, and the analysis of the FoxO3a p53 is induced by several types of stimuli that are non- or PUMA sequences was performed with a T7E1 (T7 genotoxic, including serum starvation21, ischemia/reper- endonuclease) assay and western blotting. fusion22, STS23, glucocorticoids24, and TNF-α25. Various transcription factors, including p6526, Forkhead Box O3a MTS assay (FoxO3a)27, and p7328, have been indicated in PUMA- Cells were seeded (1 × 104 cells per well) in 96-well mediated, p53-independent induction of apoptosis. plates and treated with different agents for 3 days. Cell In this study, we examined the mechanism by which viability was evaluated with 3-(4,5-dimethylthiazol-2-yl)- copanlisib induces PUMA and its function in chemo- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra- sensitization and apoptosis. We further assessed the roles zolium (MTS) assay (Promega) according to the provided of FoxO3a and PUMA in the anticancer activity of instructions. Chemiluminescence was measured using copanlisib. The results present new insights into the Perkin Elmer’s Wallac Victor 1420 Multilabel Counter. mechanism of the response to copanlisib treatment, with All the assays were carried out in triplicate and repeated potentially broad implications for future applications. three times.

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Western blotting prior to copanlisib treatment, siRNA knockdown was Western blotting was performed as described in a pre- carried out using FoxO3a-specific siRNA (400 pmol) or vious study29,30. The extraction of the total proteins from the scrambled, control siRNA from Dharmacon. cells was carried out using a Nuclear and Cytoplasmic Protein Extraction kit, and the protein concentration was Mitochondrial membrane potential (MMP) assay determined with an Enhanced BCA Protein Assay kit. The The MMP was analyzed by JC-1 staining according to lysates were diluted to 5 µg/µL, and a ten percent SDS- the manufacturer’s instructions. Briefly, WT or PUMA- PAGE gel was used to resolve 60 µg of each of the protein KO HCT116 cells were seeded in 12-well plates and samples. The proteins were transferred to membranes treated with copanlisib for 24 h. The cells were then made of PVDF from EMD Millipore at 135 mA and washed with PBS and suspended in fresh medium. The 100 mV on a Bio-Rad Trans-Blot from Bio-Rad Labora- cells were then incubated with 5,5′,6,6′-tetrachloro- tories, Inc. After blocking the membranes in five percent 1,1′,3,3′-tetraethylbenzimidazolcarboc yanine iodide (JC- skim milk at room temperature for 60 min, the mem- 1) staining solution (5 μM) at 37 °C and 5% CO2 for branes were incubated in 5% BSA solution containing 20 min. After washing with PBS, the fluorescence intensity primary antibodies at 4 °C overnight. Then, after washing was visualized using a confocal microscope (Carl Zeiss, the membranes four times with TBS-Tween for 10 min Germany). The red/green fluorescence intensity ratio was each, the membranes were incubated at room tempera- analyzed by the ImageJ software. ture with secondary horseradish peroxidase (HRP)-conjugated antibodies for 60 min. The membranes were Luciferase activity assay washed with TBS-Tween six times, and the signals were A genomic wild-type (WT) fragment of the first intron visualized using an ECL reagent from EMD Millipore on of PUMA containing two FoxO3a sites was cloned into X-ray films. The primary antibodies were as follows: the pBV-Luc vector to generate the PUMA luciferase cleaved caspase-3 (#9661, Cell Signaling Technology), reporter construct as previously described1. Next, the site- cleaved caspase 8 (#9748, Cell Signaling Technology), directed mutagenesis QuickChange XL kit (Stratagene, cleaved caspase-9 (#9505, Cell Signaling Technology), USA) was used to introduce mutations into the FoxO3a PUMA (#4976, Cell Signaling Technology), Bim (#2819, binding sites. To assess the reporter activity, cells were Cell Signaling Technology), Mcl-1 (#4572, Cell Signaling transfected with the reporter harboring WT or mutant Technology), Bid (#2002, Cell Signaling Technology), Bax PUMA along with the β-galactosidase reporter pCMVβ (#2772, Cell Signaling Technology), Bcl-xL (#2764, Cell (Promega, Madison, WI, USA) as the transfection control. Signaling Technology), Bcl-2 (#4223, Cell Signaling The luciferase activity was measured after collecting the Technology), p-AKT (#4060, Cell Signaling Technology), cell lysates. Each reporter assay was carried out in tripli- AKT (#9272, Cell Signaling Technology), p-mTOR cate and repeated three times. (#2974, Cell Signaling Technology), mTOR (#2972, Cell Signaling Technology), cytochrome c (#4272, Cell Sig- Chromatin immunoprecipitation (ChIP) naling Technology), Noxa (sc-515840, Santa Cruz Bio- ChIP was performed using the ChIP Assay kit from technology), Cox IV (ab14744, Abcam), p-FoxO3a Upstate Biotechnology (Lake Placid, NY, USA) as pre- (ab240127, Abcam), FoxO3a (ab109629, Abcam), and viously described, using an antibody against FoxO3a. β-actin (A5441, Sigma). Analysis of the precipitates was performed through PCR using the following primers: 5′-TGGGTGTGGCCG Real-time reverse transcriptase (RT) PCR CCCCT-3′ and 5′-GCGCACAGGTGCCTCGGC-3′. Real-time PCR was performed as previously descri- bed31,32. Total RNA was isolated using the Mini RNA Iso- Apoptosis lation II Kit from Zymo Research (Orange, CA) according Apoptosis was detected by staining the nuclei with to the provided instructions. One microgram of total RNA Hoechst 33258 from Invitrogen as previously descri- was used to synthesize cDNA using Invitrogen’sSuper- bed33,34. Staining with propidium iodide (PI)/Annexin V Script II reverse transcriptase. Real-time PCR for PUMA was carried out using PI and annexin Alexa 488 from and GAPDH was conducted as previously described31,32. Invitrogen according to the instructions. To analyze the release of cytochrome c, the cytosolic and mitochondrial Cell transfection and knockdown using siRNA fractions were isolated by differential centrifugation as The transfection of cells with Lipofectamine 3000 from instructed and probed for cytochrome c by western Invitrogen was carried out according to the manu- blotting. In the colony formation assay, the treated cells facturer’s instructions. Vectors constitutively expressing were seeded in plates (12-well) at the indicated dilutions, FoxO3aTM (FoxO3a triple mutant; Addgene), pCMV, grown for 10–14 days, and then stained with crystal violet WT, and AKT were transfected as specified. One day from Sigma.

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Fig. 1 Copanlisib induced apoptosis in CRC cells. A Indicated CRC cells were treated with an increasing dose of copanlisib for 72 h. The viability of cells was analyzed by the MTS assay. B Indicated cell lines were treated with copanlisib at the indicated concentration for 24 h. Apoptosis was analyzed by nuclear fragmentation assay. C HCT116 cells were treated with 100 nM copanlisib with or without 10 μM z-VAD-fmk pretreatment. Apoptosis was analyzed by nuclear fragmentation assay. D HCT116 cells were treated with 100 nM copanlisib for 24 h. The indicated protein level was analyzed by Western blotting. Results in B and C were expressed as means ± SD of three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001.

PDX mouse model definition of the ethical endpoint was the time point Nu/Nu mice (5–6 weeks old, female; Charles River) when the dimension of a tumor was 1.5 cm or more on and NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, any side. After dissection, the tumors were formalin- 4–6 weeks old, Jackson Laboratory) were placed in cages fixed (10%) and paraffin-embedded. Immunostaining (micro isolator) in a sterile environment and were given was performed using TUNEL (terminal deox- access to chow, and water ad libitum. Two PDX models ynucleotidyl transferase-mediated dUTP nick end were established using primary tumors resected from labeling) from EMD Millipore and active caspase-3 treatment-naïve, newly diagnosed CRC patients. The antibodies from Cell Signaling Technology. The tumor patients provided written informed consent, and the sections (paraffin-embedded, 5 μm) described above study was approved by the IRB. The tumors were sub- were incubated with secondary antibodies (AlexaFluor cutaneously implanted and passaged. Briefly, de- 488-conjugated; Invitrogen) for detection and DAPI identified patient CRC samples were delivered to the (4′,6-diamidino-2-phenylindole) to counterstain the laboratory in 1× Antibiotic/Antimycotic solution (Invi- nucleus. trogen) within 4 h after tumor resection. The tissues were cut into 25-mg pieces and directly implanted Xenograft studies subcutaneously into both flanks of NSG mice. Tumors The mice described above were subcutaneously injected passaged and expanded for two generations (P3) in NSG with WT or PUMA-KO HCT116 cells (4 × 106 cells) in mice were used for the described experiments. The mice both flanks. After seven days of tumor growth, the mice were randomly divided into two groups, and no blinding were randomly divided into two groups, and no blinding was performed. Treatment was carried out for 20 days was performed. Intraperitoneal (i.p.) injections of copan- following the procedure for cell line xenografts (n = 6 lisib (30 mg/kg) were administered for ten consecutive mice per group). Using calipers, the growth of the days. For combination studies, the mice were randomly tumors was measured, and the volumes were deter- divided into four groups, and no blinding was performed. mined using the formula 1/2 × length × width2.The I.p. injections of 10 mg/kg copanlisib and 25 mg/kg 5-FU

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Fig. 2 Upregulation of PUMA expression by copanlisib correlates with apoptosis induction in CRC cells. A HCT116 cells were treated with copanlisib at the indicated concentration for 24 h. The indicated protein level was analyzed by western blotting. B WT and p53-knockout (p53-KO) HCT116 cells were treated with 100 nM copanlisib at indicated time points. PUMA expression was analyzed by western blotting. C HCT116 cells were treated with 100 nM copanlisib at indicated time points. PUMA mRNA induction by copanlisib was analyzed by real-time reverse transcriptase (RT) PCR. D HCT116 cells were treated with 100 nM copanlisib with or without actinomycin D pretreatment. UMA expression was analyzed by western blotting. E Western blot analysis of PUMA expression in indicated CRC cell lines treated with 100 nM copanlisib for 24 h. Relative PUMA expression, which was quantiﬁed by the ImageJ program and normalized to that of β-actin, is indicated, with that in untreated cells arbitrarily set as 1.0. F Indicated CRC cell lines were treated with 100 nM copanlisib for 24 h. Apoptosis was analyzed by nuclear fragmentation assay and plotted against PUMA induction from E.

(APP Pharmaceuticals, Schaumburg, IL) were adminis- Results tered every other day or together for 10 consecutive days. Copanlisib promotes growth inhibition and apoptosis in The tumor growth and volumes were estimated as CRC cells described in the previous section. After euthanizing the To investigate the effect of copanlisib on CRC cells, we mice when the tumor volumes reached ~1 cm3, the same first examined the viability of the CRC cells treated for 72 procedure for fixation, labeling, immunostaining, and h with increasing concentrations of copanlisib. The MTS visualization was followed as described in the previous results demonstrated that copanlisib inhibits CRC cell section. proliferation (Fig. 1A). An in vitro assay was used to ensure the safety of the tested concentrations, and Statistical analysis copanlisib toxicity was examined in human NCM356 and For this analysis, GraphPad Prism IV software was used. NCM460 cells. However, after the treatment of these cells P-values were determined by Student’s t-test, and a value with similar concentrations of copanlisib, there was no <0.05 was deemed significant. The mean ± one SD (stan- major difference in its effects on cell viability (Fig. 1A). It dard deviation) is presented in the figures. was clear from the data that copanlisib had no effect on

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Fig. 3 PUMA-mediated copanlisib-induced apoptosis. A WT and PUMA-KO HCT116 cells were treated with copanlisib at indicated concentrations for 24 h. Apoptosis was analyzed by nuclear fragmentation assay. B WT and PUMA-KO DLD1 cells were treated with copanlisib at indicated concentrations for 24 h. Apoptosis was analyzed by nuclear fragmentation assay. C WT and PUMA-KO HCT116 or DLD1 cells were treated with 100 nM copanlisib for 24 h. Apoptosis was analyzed by flow cytometry. D WT and PUMA-KO HCT116 cells were treated with 100 nM copanlisib for 24 h. The indicated protein level was analyzed by western blotting. E Cytosolic fractions isolated from WT and PUMA-KO HCT116 cells treated with 100 nM copanlisib for 24 h were probed for cytochrome c by western blotting. β-actin and cytochrome oxidase subunit IV (Cox IV), which are expressed in cytoplasm and mitochondria, respectively, were analyzed as the control for loading and fractionation. F Colony formation of WT and PUMA-KO HCT116 cells treated with 100 nM copanlisib for 48 h following crystal violet staining of attached cells at 14 days. Left, representative pictures of colonies; Right, quantification of colony numbers. Results in A–C and F were expressed as means ± SD of three independent experiments. *P < 0.05, **P < 0.01; ***P < 0.001. nontransformed cells but had a selective inhibitory effect Therefore, our results indicate that copanlisib-induced on CRC cells. The pro-apoptotic effect of copanlisib was caspase-dependent apoptosis in CRC cells. examined using a nuclear fragmentation assay. Our results showed that copanlisib treatment significantly PUMA expression is induced by copanlisib in a p53- increased the apoptotic rates of DLD1 and HCT116 cells independent manner (Fig. 1B). Consequently, to prove that cell death was the Next, we evaluated the impact of copanlisib on proteins result of caspase-dependent apoptosis, we pretreated the of the Bcl-2 family and observed the induction of PUMA cells with z-VAD-fmk, a pan-caspase inhibitor. Our after treatment with copanlisib (Figs. 2A and Supple- findings demonstrated that the pro-apoptotic effects of mentary Fig. S1A). In both WT and p53-depleted copanlisib on the DLD1 cells and HCT116 cells were HCT116 cells, copanlisib-induced PUMA upregulation in reversed by the pan-caspase inhibitor z-VAD-fmk (Fig. a time-dependent manner (Fig. 2B). However, the lack of 1C). Subsequently, treatment with copanlisib increased p53 slightly delayed the copanlisib-induced PUMA the levels of cleaved caspase-3, -8, and -9 (Fig. 1D). induction (Fig. 2B). Furthermore, we observed the peak

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Fig. 4 (See legend on next page.)

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(see figure on previous page) Fig. 4 AKT/FoxO3a axis-mediated copanlisib-induced PUMA upregulation. A HCT116 cells were transfected with FoxO3a for 24 h. The indicated protein level was analyzed by western blotting. B WT and p53-KO HCT116 cells were transfected with either a control scrambled siRNA or a FoxO3a siRNA for 24 h, and then treated with 100 nM copanlisib for 24 h. The indicated protein level was analyzed by western blotting. C Schematic representation of the genomic structure of PUMA highlighting the two FoxO3a binding sites (AAACA) within the first intron. Asterisks indicate binding site mutations. D p53-KO HCT116 cells were transfected with a reporter plasmid containing the WT or mutant FoxO3a binding sites in the PUMA promoter, along with a FoxO3a triple mutant (FoxO3aTM) expression construct or the control empty vector. The reporter activities were measured by luciferase assay 24 h later. E p53-KO HCT116 cells were transfected with the reporter containing the WT or mutant FoxO3a binding sites for 24 h, and then treated with 100 nM copanlisib for 24 h. Luciferase activity was quantified. F Chromatin immunoprecipitation (ChIP) was performed on fixed p53-KO HCT116 cells following 6 h 100 nM copanlisib treatment. An antibody specific for FoxO3a was used to show specificity. PCR was carried out using primers surrounding the FoxO3a binding sites in the PUMA promoter. G HCT116 cells were treated with 100 nM copanlisib at indicated time points. The indicated protein level was analyzed by western blotting. H HCT116 cells were transfected for 24 h with WT, constitutively active mutant AKT, or control empty vector. The indicated protein level was analyzed by western blotting. I HCT116 cells were transfected for 24 h with constitutively active mutant AKT or control empty vector and then were treated with 100 nM copanlisib for 24 h. The indicated protein level was analyzed by western blotting. J HCT116 cells were transfected with either a control scrambled siRNA or a FoxO3a siRNA for 24 h, and then treated with 100 nM copanlisib for 24 h. The indicated protein level was analyzed by western blotting. Results in D and E were expressed as means ± SD of three independent experiments. **P < 0.01.

level of PUMA mRNA induction within eight hours and the ΔΨm in the copanlisib-treated WT and PUMA-KO that of PUMA protein induction within 24 h of copanlisib HCT116 cells by JC-1 staining. Our results indicated that treatment (Fig. 2C). This induction could be attenuated by the copanlisib-induced loss of the ΔΨm was absent in the actinomycin D, a transcription inhibitor (Fig. 2D). These PUMA-KO cells (Supplementary Fig. S2A, B). In addition, results indicate that PUMA expression is induced by in a long-term clonogenic assay, significantly enhanced copanlisib through a transcription mechanism that is resistance to copanlisib was observed in the PUMA-defi- independent of p53. A correlation between copanlisib- cient cells compared with the WT cells (Fig. 3F). Hence, mediated PUMA induction and apoptosis was also these results suggest the necessity of PUMA for the revealed in 12 CRC cell lines (Fig. 2E). After treatment, induction of apoptosis by copanlisib in CRC cells. the cell lines with endogenously low PUMA expression but strongly induced PUMA expression exhibited high Activation of PUMA transcription by FoxO3a after apoptotic levels, while the cell lines with little or no copanlisib treatment induction of PUMA expression had barely detectable or FoxO3a, a transcription factor, can induce PUMA low levels of apoptosis (Fig. 2F). Therefore, PUMA expression in mast cells and T cells after cytokine depri- expression selectively induced by copanlisib treatment vation35. Copanlisib has been implicated in AKT inhibi- enhances apoptosis in CRC cells. tion36, which leads to FoxO3a phosphorylation and arrests its nuclear localization; therefore, we assessed the PUMA is required for copanlisib-induced apoptosis roles of FoxO3a and AKT in copanlisib-mediated induc- We examined the effect of copanlisib on PUMA-KO tion of PUMA expression. We observed that in the p53- HCT116 cells. Apoptosis was observed in WT cells in a KO cells, the expression of FoxO3aTM, a constitutively dose-dependent manner, with over 30% apoptotic cells active mutant of FoxO3a, induced PUMA upregulation after 100 nM copanlisib treatment (Fig. 3A). Remarkably, (Fig. 4A). In the WT and p53-KO HCT116 cells, siRNA- the apoptosis induced by copanlisib was nearly absolutely mediated depletion of FoxO3a abrogated the copanlisib- abolished in PUMA-KO cells (Fig. 3A). Such a require- mediated induction of PUMA (Fig. 4B), indicating that ment for PUMA was not cell line-specific, as copanlisib FoxO3a is required for PUMA induction following kinase prevented the apoptosis of the previously described inhibition. PUMA-KO DLD1 CRC cells (Fig. 3B). This decline in The mouse and human PUMA promoters possess a apoptosis in PUMA-deficient cells was confirmed by conserved FoxO binding element (AAACA)37. We noted further analysis using annexin V/PI staining (Fig. 3C). The that in addition to this site, two bases apart, another deficiency in PUMA attenuated copanlisib-induced cas- FoxO3a binding element exists in the reverse orientation pase-9 and -3 activation (Fig. 3D). Interestingly, a higher in the first intron of PUMA (Fig. 4C). To examine the role baseline level of cleaved caspase-9 was observed in the of these binding sites in the induction of PUMA expres- PUMA-KO HCT116 cells (Fig. 3D). We also found that sion, a 1.1-kb fragment harboring these two sites was PUMA depletion blocked the copanlisib-mediated cyto- cloned along with luciferase reporters and analyzed. chrome c release into the cytosol in HCT116 cells (Fig. Furthermore, the mutation of these binding sites was also 3E). To investigate the role of mitochondrial dysfunction carried out to determine their precise function in the in copanlisib-induced apoptosis, we analyzed the loss of induction of PUMA expression (Fig. 4C). FoxO3aTM

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Fig. 5 Copanlisib suppresses the growth of patient-derived tumors. A Tumor volume quantiﬁcation of established PDX1 tumors in NSG mice treated with vehicle or copanlisib. B Kaplan–Meier survival curve of PDX1 tumor-bearing mice from treatment start time in vehicle and copanlisib treatment groups. C Mice with PDX1 tumors were treated with 30 mg/kg regorafenib or the vehicle as in A for four consecutive days. PUMA level in vehicle-treated and copanlisib-treated PDX1 tumor samples was analyzed by western blotting. D Real-time PCR analysis of PUMA in tumor samples from mice treated as in C. E Cleaved caspase-3 immunostaining of PDX1 tumors samples from mice treated as in C. Scale bars: 25 μm. F TUNEL immunostaining of PDX1 tumors samples from mice treated as in C. Scale bars: 25 μm. G Body weight of tumor-bearing mice from treatment start time in vehicle and copanlisib treatment groups. Results in A and D–F were expressed as means ± SD of three independent experiments. **P < 0.01. transfection or copanlisib treatment of cells led to (Fig. 4G), which prevented its nuclear translocation and remarkable activation of the WT PUMA reporter. In subsequent transactivation. In contrast, phosphorylation comparison, the mutations in the binding sites eliminated of FoxO3a by the transfection of either WT or con- the responsiveness of the PUMA reporter to FoxO3aTM stitutively active AKT suppressed the baseline expression and abolished the effects of copanlisib (Fig. 4D, E). In the of PUMA (Fig. 4H) and limited the induction of PUMA by ChIP assay, we observed that the binding of FoxO3a to the copanlisib (Fig. 4I). In addition, the siRNA-mediated PUMA promoter was markedly increased following transient knockdown of FoxO3a led to the suppression of treatment with any one of the agents (Fig. 4F). Thus, the PUMA induction and apoptosis induced by copanlisib according to the abovementioned results, the direct (Fig. 4J). Thus, PUMA induction by FoxO3a after treat- binding of FoxO3a to the PUMA promoter activates its ment with copanlisib was possibly mediated via inhibition transcription after treatment with copanlisib. of AKT.

Inhibition of AKT drives induction of PUMA by copanlisib Suppression of PDX model growth by copanlisib Then, we examined the effect of copanlisib on PUMA For in vivo examination of the effects of copanlisib, we expression, and the role of AKT, an antiapoptotic kinase used PDX models, which mimic the histopathology and that is characterized by abnormal activation in cancer heterogeneity of patient tumors and have been used to cells. Treatment with copanlisib markedly inhibited the predict clinical responses of patients to particular drugs. phosphorylation of AKT at S473; this effect was con- Mice harboring PDX1 tumors were arbitrarily grouped current with the decreased phosphorylation of FoxO3a and subjected to daily treatment with either copanlisib or

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vehicle control. Potent inhibition of PDX1 tumor growth similar results were not observed in PUMA-KO cells (Fig. and an increase in survival time of the tumor-bearing 7G). Copanlisib combined with regorafenib, which indu- mice due to copanlisib were observed (Fig. 5A, B). Ana- ces PUMA expression through the GSK-3β/NF-κB- lyses of the RNA and protein from the tumor tissues mediated pathway39, also revealed an increased induction confirmed the induction of PUMA by copanlisib (Fig. 5C, of apoptosis that was dependent on PUMA (Fig. 7F, H). D). Furthermore, an enhanced number of TUNEL- Therefore, the data suggest that PUMA regulates apop- positive cells and enhanced staining of cleaved caspase-3 tosis by mediating the chemosensitization activity of after 20 days of treatment were observed (Fig. 5E, F). In copanlisib and thus enhances the therapeutic efficacy of addition, there was a similar response of PDX2 tumors to copanlisib. copanlisib (Supplementary Fig. S3A–F). Thus, copanlisib The same outcomes were observed in vivo when nude promoted apoptosis and impaired proliferation. Upon mice bearing WT or PUMA-KO HCT116 xenograft treatment of the tumor-bearing mice with copanlisib, tumors were treated with copanlisib (10 mg/kg), 5-FU there was no significant change in body weight change, (25 mg/kg), or the combination. The growth of the WT and the mice did not show any obvious signs of toxicity tumors was more effectively suppressed by the combina- (Fig. 5G and Supplementary Fig. S3G). tion treatment than with either the copanlisib or 5-FU treatment alone (Fig. 7I). However, this enhanced tumor The antitumor activity of copanlisib in a xenograft model is suppression was significantly abolished in the PUMA-KO supported by PUMA tumors (Fig. 7I), which also correlated with a decline in To study the mechanism, nude mice were injected with apoptosis as detected by active caspase-3 staining (Fig. 7J) WT or PUMA-KO HCT116 cells to establish a tumor and TUNEL staining (Fig. 7K). Thus, the in vitro and xenograft model, and these mice were then treated with in vivo chemosensitization effects of copanlisib are copanlisib for 5 days. The assessments were performed as mediated by PUMA, and further manipulation may described in the methods section. WT tumors responded enhance the treatment efficacy of copanlisib. to the treatment by exhibiting slower growth, and the size of the treated tumors was nearly 20% that of the untreated Discussion tumors on day 22 (Fig. 6A). In contrast, PUMA-KO The observations of this study provide insight into the tumors were less sensitive to copanlisib treatment (Fig. mechanism of the anticancer activity of copanlisib. Several 6A). Furthermore, phosphorylation of AKT and FoxO3a previous studies have examined the effects of copanlisib was suppressed after treatment (Fig. 6B), and the on cell cycle checkpoints, which are generally considered expression of PUMA was induced in the WT tumors (Fig. to be the primary mode of action in the treatment of 6B). There was a significant induction of apoptosis in the cancer40,41. Reports also suggest that copanlisib potenti- WT tumors, and TUNEL and active caspase-3 staining ates apoptosis through the mitochondrial pathway42, revealed that this induction was nearly absolutely abol- although the precise mechanism of action is still unclear, ished in the PUMA-KO tumors (Fig. 6C, D). Therefore, and the status of apoptosis as a response to copanlisib PUMA is an important factor for the in vivo apoptotic and treatment remains a speculation. The results of our study antitumor effects of copanlisib. show, for the first time, that copanlisib causes the induction of PUMA by FoxO3a after inhibition of AKT. Chemosensitization effects of copanlisib are mediated by PUMA induction contributes to most, if not all, of the PUMA observed in vivo and in vitro effects of copanlisib against Copanlisib is generally combined with conventional CRC cells, and this role of PUMA was independent of the cytotoxic treatments for cancer38. We hypothesized that p53 status and genetic background. In addition to PUMA, the chemosensitivity to copanlisib is mediated by PUMA other proteins of the BH3-only family, such as Bim, may induction due to the obvious activation of PUMA by participate in the effect of copanlisib on other types of copanlisib and other drugs. In particular, robust syner- tumors. gistic effects of copanlisib and 5-FU or regorafenib in PUMA, a member of the BH3-only Bcl-2 family, can be HCT116 cells were observed (Fig. 7A–D). We further induced by other apoptotic signals or p5317. When over- validated these findings in the DLD1 cell line (Supple- expressed in various cell lines, PUMA induces cell death mentary Fig. S4A–D). Indeed, copanlisib in association by associating with the mitochondria, and an important with 5-FU induced PUMA to a level much higher than requisite of its apoptotic activity is an intact BH3 that induced by 5-FU or copanlisib alone (Fig. 7E). This is domain43. These studies, and the findings from a tumor consistent with PUMA induction through pathways that cell line with a somatic knockout, indicate an important are both dependent and independent of p53. Therefore, role of PUMA as an in vivo apoptotic regulator44. PUMA after the combination treatment, the WT HCT116 cells has been indicated to participate in the important pro- exhibited a significantly higher degree of apoptosis, but cesses of tumorigenesis44. PUMA may be a possible target

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Fig. 6 The antitumor effects of copanlisib in vivo are PUMA-dependent. A Nude mice were injected s.c. with 5 × 106 WT or PUMA-KO HCT116 cells. After 7 days, mice were treated with 30 mg/kg copanlisib or the vehicle control for 10 consecutive days. Tumor volume at indicated time points after treatment was calculated and plotted with P-values, n = 6 in each group. B Mice with WT HCT116 xenograft tumors were treated with 30 mg/kg regorafenib or the vehicle as in A for four consecutive days. The indicated protein level was analyzed by western blotting. C Paraffin-embedded sections of WT or PUMA-KO tumor tissues from mice treated as in B were analyzed by active caspase-3 staining. Upper, representative staining pictures; lower, active caspase-3-positive cells were counted and plotted. Scale bars: 25 μm. D Tissue sections from B were analyzed by TUNEL staining. Upper, representative TUNEL staining pictures; lower, TUNEL-positive cells were counted and plotted. Scale bars: 25 μm. Results in C and D were expressed as means ± SD of three independent experiments. **P < 0.01. for chemotherapy, as activation of PUMA inhibits the Bcl-2 increased due to copanlisib. The cytotoxic effect of growth of tumors by reinitiating apoptosis in cancer copanlisib was attenuated by knockdown of PUMA using cells45. The induction of PUMA is possible in a manner specific siRNAs, indicating the possible association of the that is independent of p53. In nongenotoxic stimulations, function of copanlisib with the p53-independent induc- such as growth factor deprivation, inflammatory cyto- tion of PUMA. kines, and kinase inhibitors, the p53-independent induc- There are few recent reports on the FoxO3a-mediated tion PUMA can be mediated by transcription factors, such induction of PUMA via suppression of the PI3K/AKT as FoxO3a, E2F1, NF-κB, and p7339. After its induction in signaling pathway27. For instance, sunitinib, a multikinase cancer cells, PUMA causes high rates of apoptosis by inhibitor, can induce the transcription of PUMA in acting on other proteins, such as Bax and Bcl-2 of the Bcl- human colorectal cancer through the AKT/FoxO3a axis47. 2 family, and triggers the caspase cascade46. In this study, In response to the deprivation of cytokines or growth we observed a marked induction of PUMA expression by factors, PUMA expression is directly upregulated by copanlisib in a time- and dose-dependent manner in p53- FoxO3a when PI3K/AKT signaling is blocked47. AKT null cells and WT cells. Subsequently, the ratio of Bax/ activation can be suppressed by copanlisib; hence, it is

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Fig. 7 PUMA mediates the chemosensitization effects of copanlisib in vitro and in vivo. A HCT116 cells treated with copanlisib, 5-FU, or their combination at indicated concentrations. The viability of cells was analyzed by the MTS assay. B Combination index was calculated form (A). C HCT116 cells treated with copanlisib, Regorafenib, or their combination at indicated concentrations. The viability of cells was analyzed by the MTS assay. D Combination index was calculated form (C). E HCT116 was treated with 10 nM copanlisib, 5 μg/mL 5-FU, or their combination for 24 h. The indicated protein level was analyzed by western blotting. F HCT116 were treated with 10 nM copanlisib, 0.5 μM Regorafenib, or their combination for 24 h. The indicated protein level was analyzed by western blotting. G WT and PUMA-KO HCT116 were treated with 10 nM copanlisib, 5 μg/mL 5-FU, or their combination for 24 h. Apoptosis was analyzed by nuclear fragmentation assay. H WT and PUMA-KO HCT116 were treated with 10 nM copanlisib, 0.5 μM Regorafenib, or their combination for 24 h. Apoptosis was analyzed by nuclear fragmentation assay. I Nude mice were injected s.c. with 5 × 106 WT or PUMA-KO HCT116 cells. After 7 days, mice were treated with 10 mg/kg copanlisib daily, 25 mg/kg 5-FU every other day by i.p. injection, or their combination for ten consecutive days. Tumor volume at indicated time points after treatment was calculated and plotted with P-values for indicated comparisons, n = 6 in each group. J Mice with WT or PUMA-KO HCT116 xenograft tumors were treated with 10 mg/kg regorafenib daily, 25 mg/kg 5-FU every other day, or their combination as in I for four consecutive days. Parafﬁn-embedded sections were analyzed by active caspase-3 staining. Active caspase-3-positive cells were counted and plotted. K Tissue sections from (I) were analyzed by TUNEL staining. TUNEL-positive cells were counted and plotted. Results in G–I and K were expressed as means ± SD of three independent experiments. **P < 0.01.

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probable that the inhibition of AKT/FoxO3a signaling by Publisher’s note copanlisib may cause the induction of PUMA. Concurrent Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. FoxO3a knockdown weakened the effect of copanlisib on 37 the induction of PUMA . Therefore, the inhibition of Supplementary Information accompanies this paper at (https://doi.org/ AKT/FoxO3a signaling may participate in the copanlisib- 10.1038/s41419-020-03154-w). mediated, p53-independent induction of PUMA. Received: 30 May 2020 Revised: 13 October 2020 Accepted: 15 October The dose of copanlisib used in this study is similar to 2020 that used in earlier studies on apoptosis36. Our results indicate the potential role of PUMA induction in chemosensitization by copanlisib. Thus, combinations of different classes of PUMA-inducing agents may facilitate References the simultaneous induction of PUMA through multiple 1. Knickelbein, K. et al. 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