Dual HDAC1/2 and PI3K/AKT pathway inhibition by fimepinostat led to robust tumor growth inhibition in both ARPC and NEPC models including cell line– and patient-derived xenografts. HDAC1/2 inhibition combined with PI3K/AKT inhibition was more effective than targeting each pathway alone, producing growth inhibitory effects through cell-cycle inhibition and apoptosis. Molecular profiling revealed on-target effects of combined HDAC1/2 and PI3K/AKT inhibition independent of tumor phenotype. Fimepinostat therapy was also associated with the suppression of lineage transcription factors including AR in ARPC and Achaete-scute homolog 1 (ASCL1) in NEPC. Together, these results indicate that fimepinostat represents a novel therapeutic that may be effective against both ARPC and NEPC through CRPC subtype-dependent and -independent mechanisms.Castration-resistant prostate cancer (CRPC) consists of multiple phenotypic subtypes including androgen receptor (AR)-active prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). Tumor cells with these phenotypes can coexist between metastases within a patient and within an individual tumor. Treatments that are effective across CRPC subtypes are currently lacking. Histone deacetylation is crucial for the regulation of chromatin structure and maintenance of cancer cell state and activation of the PI3K/AKT/mTOR signaling cascade is a tumor growth–promoting pathway. We therefore investigated combined targeting of histone deacetylase (HDAC) and PI3K using a rationally designed dual inhibitor, fimepinostat, in CRPC subtypes CRPC is a heterogeneous disease constituting multiple phenotypic subtypes that often co-occur within tumors or across metastases in patients. Existing targeted therapies for CRPC do not take this into account. Here we show that fimepinostat, a dual HDAC1/2 and PI3K/AKT inhibitor investigated clinically in other cancer types but not prostate cancer, may overcome this heterogeneity by effectively inhibiting both ARPC and NEPC subtypes of CRPC. When initially diagnosed, the vast majority of metastatic prostate cancers exhibit a phenotype consistent with a secretory luminal cell that is regulated by androgens through the androgen receptor (AR) signaling program. Inhibiting the AR signaling pathway through androgen deprivation therapy (ADT) produces exceptional clinical responses in the majority of patients. However, resistance to ADT is universal, leading to a clinical state termed castration-resistant prostate cancer  and PI3K/AKT activities can exploit these vulnerabilities in treatment-resistant AR-positive CRPC and AR-negative NEPC tumor phenotypes. We evaluated romidepsin an FDA-approved HDAC1/2 inhibitor used in the treatment of T-cell lymphoma, and ipatasertib, an AKT inhibitor that has shown preliminary efficacy in clinical trials in patients with CRPC . While t2 incubators (Thermo Fisher Scientific). C4-2B , 22Rv1 cells , VCaP , LNCaP , LNCaP95 , RRID:CVCL_ZC87), LAPC4 , PC3 , and DU145  cell lines were maintained in RPMI1640 Media or DMEM  with 10% FBS  or 10% charcoal-stripped FBS (Thermo Fisher Scientific). RWPE-1  and HPrEC  were cultured in Keratinocyte Serum Free Medium (Thermo Fisher Scientific) supplemented with 0.05 mg/mL bovine pituitary extract and 5 ng/mL human epidermal growth factor. NCI-H660 , MSKCC EF1 , and LASCPC-01  cell lines were maintained in HITES media (ATCC) with 5% FBS . Short tandem repeat (STR) profiling of cell lines was performed on a yearly basis and Mycoplasma testing was performed twice yearly. To further expand NCI-H660 cell numbers, NCI-H660 cells were injected subcutaneously in immune compromised male mice as described previously  tumors  were seeded in 96-well plates and treated with increasing concentrations of romidepsin or a control HDAC inhibitor (trichostatin A) for 1 hour. An equal volume of HDAC-Glo I/II reagent was added to the wells and luminescence measured on a GENios plate reader (Tecan).The HDAC-Glo I/II activity assay (Promega) was used to measure HDAC activity as per the manufacturer's instructions. Briefly, cells  and secondary antibodies. Proteins were visualized using Clarity Western enhanced chemiluminescence Substrate (Bio-Rad) or Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore) and a ChemiDoc imaging system (Bio-Rad).For tissue slice culture, formalin-fixed and paraffin-embedded tissue sections on positively charged glass slides were incubated at 65°C for 1 hour, placed in xylene for 5 minutes to deparaffinize the sections, and rehydrated with successive washes for 5 minutes each in 100%, 95%, and 70% ethanol. Antigen retrieval was performed by boiling the slides in 0.2 mol/L citric acid and 0.2 mol/L sodium citrate in a pressure cooker. Tissue sections were washed with PBS, blocked with 2.5% horse serum for 1 hour, and incubated with primary antibody  diluted in 2.5% horse serum for 12–18 hours at 4°C. Endogenous peroxidases were blocked with hydrogen peroxide. Tissue sections were washed with PBS and incubated with secondary ImmPRESS-HRP anti-mouse or anti-rabbit IgG antibodies (Vector Laboratories) for 1 hour at room temperature. Staining was visualized with DAB peroxidase substrate (Dako) and quenched in water. Tissue sections were counterstained with hematoxylin prior to dehydration with successive washes for 5 minutes in 70%, 95%, and 100% ethanol and xylene followed by mounting with a glass coverslip using VECTASHIELD Antifade Mounting media (Vector Laboratories). Digital slide images were captured on a VENTANA DP 200 slide scanner (Roche) and quantification of average nuclear density was performed using QuPath-0.2.3 and ImageJ Fiji  on the VENTANA Discovery Ultra  autostainer. Onboard deparaffinization was conducted in DISCOVERY Wash buffer . Subsequent heat-induced epitope retrieval was performed in DISCOVERY CC1 solution . TMA sections were incubated with AR monoclonal rabbit antibody  and synaptophysin rabbit monoclonal antibody  at a dilution of 1:100 and 1:50, respectively in Ventana Antibody Diluent with Casein . The primary antibody was bound with DISCOVERY anti-Rabbit HQ secondary antibody , followed by DISCOVERY Anti-HQ HRP  enzyme conjugate. To further increase signal intensity, tyramide amplification was achieved with the DISCOVERY HQ Amp kit  followed by DISCOVERY Amp Anti-HQ HRP . The antibody complex was visualized using the ChromoMap DAB detection Kit . Hematoxylin II  and Bluing Reagent  were used to counterstain the sections. Slides were scanned at a 40X magnification using the Ventana DP 200 instrument (VMSI) and visualized with HALO (Indica Labs).Paired-end whole-exome and whole-genome next-generation sequencing was performed using Illumina HiSeq 2500 on genomic DNA isolated from PDX tissues. Paired germline genomic DNA was isolated from PDX tissue donor's blood. Sequence reads were aligned to the human reference genome hg38 using the Burrows-Wheeler Aligner-mem tool  from public data portals .22Rv1 and C4-2B cell line exome sequence data are analyzed by adopting tumor-only pipeline of calling mutation and copy-number determination following standard BWA alignment to HG38 reference genome and GATK good practices steps. A tumor-only analysis is unfavorable for accurate ploidy estimation, so we avoided reporting a ploidy estimates of these cell lines. Because the NCI-H660 exome sequence data were unavailable, we retrieved the desired genes’ genotype calls  was used to determine the Loewe synergy score  through accession number GSE223758. All other data and material will be made available upon request.50s of approximately 100 nmol/L, indicating preferential antiproliferative activity in the malignant state. We examined the temporal onset of markers of cell death in the 22Rv1 and LNCaP95 prostate cancer cell lines upon treatment with fimepinostat. Immunoblot analyses showed clear evidence of cleaved PARP and cleaved caspase-3 as early as 36 hours after treatment initiation (We and others have demonstrated that metastatic CRPC (mCRPC) exhibit a substantial degree of heterogeneity, with tumors comprising ARPC features coexisting in the same patient with tumors exhibiting NEPC characteristics ; ref. 2.itiation .HDACs are established as being required for AR function in prostate cancer and HDAC inhibition can lower AR protein levels by suppressing AR mRNA transcription . Further50 of quisinostat (8.36 nmol/L) was similar to fimepinostat (1.19 nmol/L) but significantly lower than vorinostat (953.8 nmol/L) in the 22Rv1 cell line  for 48 hours. Immunoblot analysis showed a mild reduction of AR and Myc levels in both cell line models with intermittent treatment at doses of 30 and 100 nmol/L  and S2D.We next evaluated whether the modulation of Myc and AR levels after fimepinostat treatment occurs via transcriptional or posttranscriptional mechanisms. Quantitative PCR was performed on 22Rv1 and LNCaP95 cells treated over a time course with fimepinostat using primers specifically designed to quantify AR-FL mRNA, AR-V7 mRNA, AR pre-mRNA, Myc mRNA, and Myc pre-mRNA. Our data indicated a rapid reduction in AR and Myc pre-mRNA levels beginning within 1–2 hours of fimepinostat treatment followed thereafter by a reduction in AR-FL, AR-V7, and Myc mRNA levels  and B. AOn the basis of these findings, we anticipated that AR splice variant and structural variant expression should be inhibited in prostate cancer with fimepinostat treatment as the primary mode of AR inhibition is transcriptional suppression. We tested 22Rv1 and LNCaP95 cell lines that were stably engineered to express doxycycline-inducible shRNAs against GFP (control), AR exon 8, AR cryptic exon 3 (CE3) specific to AR-V7, and AR exon 1 for targeted expression of AR isoforms. In this setting, we found that fimepinostat treatment suppressed AR-FL and AR-Vs including AR-V7 . We also50 in response to fimepinostat when compared with 22Rv1 control cells expressing GFP  and insulinoma-associated 1 (INSM1) in the NCI-H660 cell line . However+ NEPC cell line LASCPC-01 . Residual tumor tissues were collected at the end of the experiment and IHC studies revealed reduced expression of the proliferative marker Ki-67  and LuCaP 35CR  tissue slices on day 3 with fimepinostat treatment at 300 nmol/L relative to vehicle alone  and S6B.3 ± 26.0 mm3 with fimepinostat treatment compared with 330.3 mm3 ± 72.2 mm3 with vehicle (P = 0.000031). Fimepinostat therapy did not induce any gross toxicities and mouse weights were not significantly different compared with vehicle treatment . Collectively, these data were especially notable as these xenograft models reflect a treatment-resistant disease state for which no standard or novel therapies beyond cytotoxic chemotherapy are currently approved.Fimepinostat therapy was also tested in the NCI-H660 NEPC cell line xenograft model . At the As our findings indicated a broad growth inhibitory effect of HDAC inhibition in preclinical prostate cancer models, we further explored the activities of the class I-selective HDAC inhibitors etinostat and romidepsin, class I/IV-selective HDAC inhibitor mocetinostat, and the pan-HDAC inhibitor vorinostat. Romidepsin appeared the most potent as it inhibited the cell viability of both C4-2B and NCI-H660 cells at very low-nanomolar concentrations  and S9B,Pathway analysis of RNA-seq gene expression data from ARPC and NEPC cell lines and dissociated tumor cells treated with romidepsin showed enrichment in EMT, KRAS signaling, and inflammatory signaling and de-enrichment in Myc targets, E2F targets, and G2–M checkpoint , in lineWe further evaluated the contribution of PI3K/AKT inhibition when combined with HDAC inhibition to the suppression of prostate cancer cell viability. We administered the AKT inhibitor ipatasertib, the selective PI3Kα inhibitor apelisib (BYL-719), or the selective PI3Kβ/δ inhibitor AZD8186 with romidepsin to treat the C4-2B and NCI-H660 cell lines. In each case, concurrent pharmacologic inhibition of PI3K/AKT and HDAC resulted in a more pronounced reduction in cell viability relative to single-agent inhibition . FurtherTo determine the nature of the interaction between PI3K/AKT and HDAC inhibition in prostate adenocarcinoma and NEPC cell line models, the cell viability of C4-2B and NCI-H660 lines were determined after treatment across a dose matrix with romidepsin and AZD8186, apelisib, or ipatasertib. On the basis of the Loewe additivity scores, romidepsin and AZD8186 demonstrated synergy while romidepsin and apelisib exhibited additivity in the C4-2B and NCI-H660 lines . In contPTEN, RB1, AKT1, PIK3CA, and PIK3R1 with respect to genomic alterations does not associate with fimepinostat activity in these models.PTEN encodes a dual-specificity phosphatase that functions as a direct antagonist of PI3K, a kinase involved in AKT activation. We characterized the pertinent cell lines and LuCaP PDX models and determined that none harbor PIK3CA-activating mutations . Overall−8). Of the 97 downregulated genes in common between fimepinostat and ipatasertib + romidepsin treated C4-2B cells—95 were analyzed (−8) and for TFT-associated pathways E2F_Q4_01 (4.21 e−13). These data verify the HDAC activity of fimepinostat and the impact on cell cycle through the downregulation of E2F-associated pathways. Of the 498 upregulated genes in common between fimepinostat and ipatasertib + romidepsin treated NCI-H660 cells—487 were analyzed (−10). For TFT-associated genes nuclear factor of activated T cells  and AP-1 (5.9 e−14) associated genes were significantly upregulated. The enrichment of AP-1 in the NCI-H660 cells verifies our results suggesting a role for AP-1 in fimepinostat-treated NCI-H660 cells  models and prostate cancer cell lines.Click here for additional data file.Supplementary Figure 15Supplementary Figure 15. RNA-seq volcano plots of C4-2B and NCI-H660 cells treated with vehicle, fimepinostat, ipatasertib, romidepsin, or a combination of ispatasertib + romidepsin.Click here for additional data file.Supplementary Figure 16Supplementary Figure 16. Overlapping gene expression in C4-2B and NCI-H660 cells treated with vehicle, fimepinostat, ipatasertib, romidepsin, or a combination of ipatasertib + romidepsin.Click here for additional data file.Supplementary Figure 17Supplementary Figure 17. Pathway analysis of hallmark (A) and KEGG (B) genes from C4-2B and NCI-H660 cells treated with vehicle, fimepinostat, ipatasertib, romidepsin, or a combination of ipatasertib + romidepsin.Click here for additional data file.


BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques — BH3 profiling and high-throughput kinase activity mapping — we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as   BCL2 p.G101V that reduces binding of venetoclax to BCL-2, these mutations are only observed in about half of the patients with CLL on long-term venetoclax therapy, and when these mutations do occur, they are often found at a low variant allele frequency (VAF) (The incorporation of the B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax into the treatment paradigm for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) has revolutionized the treatment of these diseases , 2. Howecy (VAF) –6. Thus,Prior studies have suggested that posttranslational modification(s) of the BCL-2 family proteins could alter their functions in governing the intrinsic apoptotic pathway. In particular, phosphorylation of antiapoptotic BCL-2 at serine 70 (S70pBCL-2) has been shown to promote cancer cell survival in response to proapoptotic stimuli and to enhance its sequestration of the proapoptotic effector BAX . PhosphoThe protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that dephosphorylates its target proteins. Numerous studies have demonstrated a tumor suppressor role of PP2A, and PP2A inactivation contributes to disease progression in several hematologic malignancies , 21–26. Here, we report data comparing venetoclax-sensitive with venetoclax-resistant malignant lymphoid cells, including a panel of cell lines, a lymphoid mouse model, and primary samples from patients with CLL before venetoclax treatment and at the time of progression while on venetoclax. We found that hyperphosphorylated BCL-2 family proteins were a crucial mechanism of intrinsic and acquired venetoclax resistance in lymphoid malignancies and we provide evidence supporting future clinical investigation of a therapeutic strategy utilizing PP2A-activating drugs (PADs) to resensitize resistant cells to venetoclax.To evaluate the potential contribution of BCL-2 family protein phosphorylation to venetoclax resistance, we examined lymphoid malignant cell lines that were intrinsically sensitive and resistant to venetoclax. We first identified 3 DLBCL cell lines — double-hit Su-DHL4, germinal center B cell TOLEDO, and activated B cell TMD8 — that were intrinsically resistant to venetoclax in relation to the intrinsically sensitive DLBCL cell line OCI-Ly1-S . We alsohttps://doi.org/10.1172/JCI170169DS1).Next, we measured the phosphorylation status of the BCL-2 family members to evaluate for an association between their phosphorylation levels and resistance to venetoclax. We observed a selective increase in T163pMCL-1 (antiapoptotic activation) and S112pBAD (proapoptotic inhibition) in intrinsically resistant Su-DHL4, TOLEDO, and TMD8 cells, and a further increase in T163pMCL-1, S70pBCL-2 (antiapoptotic activation), and S112pBAD in the acquired-resistance Su-DHL4-R and OCI-Ly1-R lines. There was, however, no consistent increase in S184pBAX (proapoptotic inhibition) . These fWe next utilized high-throughput kinase activity mapping (HT-KAM), a functional assay that detects the phosphorylation of 11 mer peptides corresponding to their protein phosphorylation sites by kinases from cell lysates and confirmed the increase in MCL-1, BCL-2, and BAD phosphorylation in both the intrinsically and acquired-resistance Su-DHL4 and OCI-Ly1-R, in comparison with OCI-Ly1-S . Importac (Cytc) release, thereby identifying the antiapoptotic protein(s) the cell functionally depends on for survival . In. Inc  or nonphosphorylatable (p.S70A) mutants of S70pBCL-2 in OCI-Ly1-S cells . We obseAlthough MCL-1 inhibition effectively kills venetoclax-resistant cell lines that are functionally dependent on MCL-1, direct targeting of MCL-1 in patients has to date proven to be challenging because of cardiotoxicity , 33. TheIncreasing doses of FTY720 at various time points showed a clear reduction of S70pBCL-2, T163pMCL-1, total MCL-1, and S184pBAX in all resistant cell lines , A and BR = 0.8633, P < 0.0001), thus suggesting that venetoclax resistance is associated with a distinct pattern of altered kinome activity. Strikingly, we observed a clear reprogramming of the kinome enzymatic activity following FTY720 treatment, reverting the resistant kinome back to a sensitive state . We also independently verified the activity of some kinases by confirming the inactivation of AKT (reduced Ser473 phosphorylation), downstream activation of GSK3β (reduced Ser9 phosphorylation) and inactivation of ERK (reduced Thr202/Tyr204 phosphorylation) in both the acquired and intrinsic venetoclax-resistant cell lines , a variation of BH3 profiling that measures the net effect of ex vivo drug treatments on the antiapoptotic dependence of cells . TreatmeTo confirm that these changes in dependencies were governed by the reduction of BCL-2 family protein phosphorylation, we induced transient overexpression of the mutants of S70pBCL-2 and T163pMCL-1 in OCI-Ly1-R and Su-DHL4 cells, respectively. Indeed, the p.S70E, but not p.S70A, mutant prevented the increase in BCL-2 dependence following FTY720 treatment, while WT MCL-1, but not the p.T163A mutant, prevented the decrease in MCL-1 dependence following FTY720 treatment . These oc release, and apoptosis, we next examined whether a mechanism underlying functional venetoclax resistance involves an altered interactome of apoptotic proteins. Indeed, we observed less effective dissociation of BAX from BCL-2 in OCI-Ly1-R or Su-DHL4 cells compared with OCI-Ly1-S when these cells were treated with venetoclax  and subsequently cotreated them with venetoclax for 24 or 48 hours. Indeed, we observed that both intrinsic and acquired-resistance lymphoid lines were sensitized to venetoclax following FTY720 pretreatment . In an uTo demonstrate that this combination is cytotoxic (rather than cytostatic), we chased this treatment combination for 6 days with a trypan blue exclusion assay using the drug combination doses with the best synergy scores and found that the combination effectively reduced live cell counts of Su-DHL4 and OCI-Ly1-R , while rTo further confirm that the cytotoxic effects of this combination require the reduction of BCL-2 family protein phosphorylation, we demonstrated that p.S70E, but not p.S70A, transfection of OCI-Ly1-R cells and WT MCL-1, but not p.T163A, transfection of Su-DHL4 cells resulted in resistance to the treatment combination, as measured by trypan blue exclusion and CellTiter-Glo (CTG) assays , F and GSince FTY720 is a PAD  and hypeAs a negative control to demonstrate that these effects were PP2A specific and involved FTY720-induced kinome enzymatic reprogramming, we showed that exposure of OCI-Ly1-R or Su-DHL4 cells to a single kinase inhibitor of MEK/ERK1/2 (PD98059) or JNK (SP600125) failed to reduce S70pBCL-2, T163pMCL1, and MCL-1, rewire cellular dependence to BCL-2, or resensitize cells to venetoclax to the degree observed with FTY720 , A and BTo assess whether our cell line findings could be recapitulated in primary CLL cells from treatment-naive patients, we used PBMCs from 28 patients with CLL with various disease characteristics , treatedTo further explore this functional mechanism of venetoclax resistance, we studied 13 paired samples from patients with CLL to compare pretreatment baseline with the time of progression while on venetoclax . CompareWe next tested whether ex vivo treatment with FTY720 could resensitize these progression samples to venetoclax. Indeed, we found that FTY720 simultaneously reduced T163pMCL-1, S70pBCL-2, S112pBAD, S184pBAX, and MCL-1 levels , rewired–/– (NSG) mice via tail-vein injection, and the mice were randomized and subjected to bioluminescence imaging (BLI) for luciferase signals on day 14. Mice without tumors were removed, while the rest were then subjected to specific treatment schedules on day 16  at the BCL-2 flexible loop, or whether the co-occurrence of S70pBCL-2 and BCL2 mutation(s) may further distort the conformation of BCL-2, leading to a further decrease in venetoclax binding.While genomic mechanisms including netoclax , 5. Moreexposure . This apOur data also suggest that venetoclax-resistant lymphoid cells may be sensitive to MCL-1 inhibition, as the resistant cells in our experiments generally depended on MCL-1 for survival. Dual targeting of both MCL-1 and BCL-2 could, in theory, overcome such a resistance mechanism; however, the development of direct MCL-1 inhibitors in the clinic has been limited by cardiotoxicity. Our findings here suggest that an alternative approach to MCL-1 inhibition that may have a better safety profile is to use PADs. In particular, we found that FTY720, a fingolimod compound that is FDA approved to treat multiple sclerosis, is a promising candidate to be studied in the clinic as a partner for venetoclax. Moreover, venetoclax is also being studied in solid tumors. For example, in breast cancer, in which BCL-2 family protein phosphorylation is implicated in tumor progression and drug resistance –52, the The significant changes we observed in the kinome enzymatic activity of venetoclax-resistant cells represent another important finding from our HT-KAM assay that supports the use of PADs. Our data suggest that targeting only 1 kinase, such as ERK1/2 or JNK, to overcome resistance may not be sufficient , A and BFinally, our work has the potential to open up new treatment approaches and to identify new therapeutic vulnerabilities for patients with lymphoid malignancies that are resistant to venetoclax. For example, the biological signatures we identified here allow the stratification of patients with lymphoid malignancies into different categories of resistance mechanisms, thus paving the way to more personalized therapeutic strategies . Coupled with the functional assays such as BH3-profiling and HT-KAM assays, our biological signatures could further assist in identifying lead compounds or repurposing FDA-approved drugs  via compound/drug library screenings based on their potential to activate PP2A/inhibit kinases, reduce BCL-2 family protein phosphorylation, and rewire survival dependencies toward BCL-2 to facilitate venetoclax-induced cell death.Additional details on methods and materials can be found in the BCL2 mutations that have been reported to affect binding to venetoclax  density gradient centrifugation method. Isolated primary cells were either immediately used for experiments or viably frozen in freezing media  for subsequent experiments. The cell lines used in this study were OCI-Ly1, Su-DHL4, TOLEDO, TMD8, Jurkat, and Stroma-NKTert. The acquired-resistance cell lines OCI-Ly1-R and Su-DHL4-R were generated from parental cells following continuous culturing in venetoclax starting from a sublethal dose of 10 nM and then doubling this dose when cells could grow at a rate similar to that of its parental cells, up to 1 μM . Acquirenetoclax . All priBH3 profiling is a functional technique that measures the proximity of cells to apoptosis and the dependence of cells on specific antiapoptotic proteins for survival. Additional details are included in the HT-KAM is a high-throughput functional platform that measures cellular kinase activity . AdditioThree different cell viability assays were used in this study: CTG, trypan blue exclusion, and annexin/Hoechst. The cell density used was approximately 0.75 million cells/mL. Additional details are included in the –/– (NSG) mice were purchased from The Jackson Laboratory. Luc-tagged OCI-Ly1-S or OCI-Ly1-R cells (1 million cells) were injected into the tail vein of 6- to 8-week-old female NSG mice, which were randomized to the treatment arms (n = 8 mice per arm). Treatments were administered until euthanization. BLI was performed for cell engraftment and tumor burden on mice using the Xenogen imager, and analysis was done using Living Image 4.2 software (PerkinElmer). Mice with no cell engraftment or tumor development were removed. Venetoclax was administered at 50 mg/kg/day p.o. , and FTY720  was administered at 4 mg/kg/day i.p.NOD/SCID/Il2Rγhttps://synergyfinder.org/#!/)  .t test, 1-way ANOVA, 2-way ANOVA, and multiple-comparison tests, with statistical significance set at a P value of less than 0.05. Pearson’s correlation coefficient R was computed for correlations between 2 variables.Statistical analyses were performed using GraphPad Prism, version 9 (GraphPad Software). Data are presented as the mean ± SD (or as otherwise specified in the figure legends) of individual values, scatter plots, heatmaps, and bar graphs with triplicates or more for cell lines and single runs for primary samples. Statistical tests included a 2-tailed Animal studies were performed under a protocol approved by the IACUC of the Dana-Farber Cancer Institute (DFCI). Patient samples were used with written informed consent and a protocol reviewed and approved by the Dana-Farber/Harvard Cancer Center IRB.All supporting data values are provided in the Supplemental SJFC and MSD conceptualized the study. SJFC, RG, CSM, JPC, and MSD designed the study methodology. SJFC, FZ, OD, RM, JXHL, LH, CER, MCC, JP, RC, YCH, MB, and DPM performed experiments. SJFC, FZ, OD, RM, JXHL, RC, YCH, MB, DPM, YCL, CSM, JPC, and MSD performed data analysis and interpretation. SJFC, LH, CER, MCC, JBI, CJW, SP, BCG, RRF, JNA, CSM, JPC, and MSD provided administrative, technical, material, or funding support. MSD supervised the study. SJFC, JPC, and MSD wrote the original draft of the manuscript. All authors reviewed and edited the manuscript.


Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib  and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by the development of resistance.We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins.PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells.The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested [CI range: 0.11–0.69] and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl−2 levels in This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination. It is estimated that by 2030 the global burden of CRC will increase by 60%, with 2.2 million new cases and one million deaths worldwide.5 Other mechanisms leading to the development of resistance include 1) Formation of new secondary site resistance mutations within the target kinase; 2) Activation of escape bypass routes involving signaling pathways such as MAPK/ERK and PI3K/AKT/mTOR; 3) Dysregulation of downstream effectors; 4) Transformation into pro-metastatic phenotypes, which enable the cancer cells to survive the effects of treatment; 5) Immune adaption within the tumor microenvironment to enable cancer cell survival, via either immune-dependent or immune-independent processes.6 Theoretically, combined therapies can produce synergistic inhibition in a relatively safe manner to reduce multiple growth signal transmission responsible for the development of drug resistance, as compared to monotherapy. There is now a growing appreciation for using combination therapeutic approaches which can be exploited through multiple modalities such as radiotherapy, chemotherapy, immunotherapy, or targeted agents.Treatment for CRC is complex and often limited by resistance to therapy, which can be intrinsic or acquired. The crosstalk between the PI3K/AKT/mTOR  and MAPK/ERK (mitogen activated protein kinase) pathways is recognized as a key mechanism of resistance to oncology therapy.RAS, BRAF, and PIK3CA arising from both pathways. There are numerous data from Phase I/II trials support the use of Palbociclib (CDK 4/6 inhibitor),7–10 Gedatolisib ,12 and PD0325901 (selective MEK1/2 inhibitor)13–15 as single agents in various types of cancers. Nonetheless, these inhibitors have limited cytoreductive effect when used as single agents because of drug resistance. As shown in breast cancer models, the combination of a CDK 4/6 inhibitor with a PI3K/mTOR inhibitor have produced synergistic treatment effects. This specific drug combination can overcome treatment-related resistance by preventing RSK activation and subsequent MAPK/ERK pathway activation.16–18 Currently, there are several active Phase I trials evaluating the combination of Palbociclib with Gedatolisib in patients with refractory malignancies including CRC. Similarly, another Phase Ib trial is evaluating the effectiveness of combining a different PI3K/mTOR dual inhibitor (Samotolisib) with a CDK4/6 inhibitor (Abemaciclib) in multiple common cancers.19 By extrapolating the available literature, we believe the approach of using Palbociclib with Gedatolisib to prevent the emergence of resistance in breast cancer is hypothetically applicable to other cancers including CRC. In parallel, there is strong preclinical evidence for the evaluation of co-inhibition using Palbociclib with PD0325901 in CRC.13–15 Of note, the combination of Gedatolisib with PD0325901 has previously been shown to have unacceptable toxicity in humans.20The PI3K/AKT/mTOR and MAPK/ERK signaling pathways are highly implicated in CRC pathogenesis with key mutations like In summary, treatment with a targeted therapy combination has multiple advantages over treatment with a single agent as anti-proliferative efficacy can be maximized within an acceptable overlapping drug toxicity limit. In comparison to monotherapy, combined drug therapies inhibit multiple-targets and have diverse cellular regulatory actions and are thus more likely to be effective in attenuating drug resistance pathways. This method has been exploited in various cancers, particularly in breast cancer. As we have noted, there is a research-gap in CRC, therefore there is a need to explore novel therapeutics using combinative drug approaches. We hypothesized that the combination of Palbociclib with either Gedatolisib or PD0325901 could produce synergistic benefits and have potential clinical relevance for the treatment of refractory CRC.50 values of single-agent Palbociclib, Gedatolisib, and PD0325901 in CRC cell lines are summarized in 50 >15 µM). LS411N and SNUC4 cells were sensitive to Palbociclib . The LS1034 cell line was relatively resistant to Gedatolisib (IC50 = 7.2 µM). All other cell lines were more sensitive to Gedatolisib with IC50 values generally within nanomolar ranges . The cell lines also had high sensitivity to PD0325901 . The Caco−2 cell line was relatively resistant to all drugs, in particular Palbociclib (IC50 >15 µM).Drug concentrations used for the combination assays and the corresponding ICKRAS mutation; CI = 0.11). The combination of Palbociclib with Gedatolisib is also synergistic in DLD−1  and Caco−2  cells. The combination of Palbociclib with Gedatolisib is minimally synergistic in the LS411N  and SNUC4  cell lines.Drug combination analysis showed that the combination of Palbociclib with Gedatolisib has a synergistic anti-proliferative effect in all CRC cell lines tested . The comThe combination Palbociclib with PD0325901 produces a synergistic growth inhibitory response in all cell lines, apart from LS411N cells  .Figure We conducted RPPA analysis using 40 primary antibodies representing multiple nodes of the PI3K/AKT/mTOR, MAPK/ERK and intracellular apoptotic signaling pathways, following 4-h treatment with Palbociclib, Gedatolisib, and their combination in the Caco−2, DLD−1, LS1034, and SNUC4 cell lines.p < .001; DLD−1 fold change= −3.85 ± 0.05, p = .012; LS1034 fold change= −2.70 ± 0.02, p < .001 and SNUC4 fold change= −2.86 ± 0.07, p < .001]. The combination Palbociclib with Gedatolisib also caused significant suppression of S6rp (S235/236) in Caco−2 , DLD−1  and a close to significant suppression in LS1034 , compared to vehicle control treated cells. The suppression of S6rp (S240/244) and S6rp (S235/236) was much more marked with the combination of Palbociclib with Gedatolisib than with either Palbociclib or Gedatolisib alone.The combination Palbociclib with Gedatolisib showed synergistic inhibition of some components of the PI3K/AKT/mTOR pathway compared to other treatment arms . The comp = .006] compared to vehicle control treated cells. In comparison to vehicle control treated cells, the combination Palbociclib with Gedatolisib appeared to exert greater suppression of phosphorylated mTOR (S448) than Palbociclib or Gedatolisib alone, especially in DLD−1 cells  and LS1034 cells . However, these changes were not statistically significant. There was no change in the levels of phosphorylated mTOR (S2481) in any of the cell lines treated with the combination Palbociclib with Gedatolisib compared to control. However, treatment with the combination Palbociclib with Gedatolisib significantly increased PTEN levels compared to control treated LS1034 cells  and Caco−2 cells .We did not observe increases in phosphorylated PDK1 (S241) or AKT (S473 and T308) with the combination Palbociclib with Gedatolisib, suggesting that there was no feedback activation of AKT signaling . In contp = .039], SNUC4  and Caco−2 cells . We also observed significantly increased Bcl−2 levels in DLD−1  and SNUC4  cells treated with the drug combination, an effect that was not observed with single-agent Palbociclib or Gedatolisib.The expression and phosphorylation of the intracellular apoptotic signaling proteins were assessed to determine if the addition of Gedatolisib can enhance the actions of Palbociclib during cell cycle progression to increase apoptosis . In compThe increase in BAX and Bcl−2 levels in DLD−1 and SNUC4 cells treated with the combination Gedatolisib with Palbociclib was not associated with an increase in caspase 3, caspase 8, cleaved caspase 7, cleaved caspase 9, and cleaved PARP levels, possibly indicating that the observed increases were not sufficient to induce total apoptosis. However, it is possible that the 4-h treatment timepoint used in our study was too early to measure the late proteomic alterations associated with apoptosis. Of interest, the addition of Gedatolisib to Palbociclib did not induce any changes in the phosphorylated Ribosomal protein (Rb (S807/811)).p < .001, DLD−1 fold change = 2.64 ± 0.49; p < .001, SNUC4 fold change = 2.39 ± 0.10; p = .001 and LS1034 fold change = 1.71 ± 0.14; p = .072. Although MAPK(T202/Y204) phosphorylation did not increase following any treatment, MEK1/2(S217/221) phosphorylation did increase after single agent and combination treatments in DLD−1 cells, as did levels of E2F1, suggesting global activation of MAPK/ERK signaling, including with the combination treatment. The fold changes for MEK1/2(S217/221) were as follows: Combination Palbociclib with Gedatolisib = 2.09 ± 0.30; p < .001, Gedatolisib = 1.81 ± 0.20; p = .003 and Palbociclib = 1.64 ± 0.19; p = .013 whilst the fold changes for E2F1 were combination Palbociclib with Gedatolisib = 1.50 ± 0.03; p < .001, Gedatolisib = 1.40 ± 0.12; p = .005 and Palbociclib = 0.94 ± 0.07; p = .845.One of the mechanisms for the development of resistance to PI3K-targeted inhibitors is the reactivation of membrane receptor tyrosine kinases (RTKs) and/or the MAPK/ERK signaling cascade. Our RPPA data showed an increase in the total EGFR in all cell lines after 4 h of treatment . Followip = .025] and SNUC4  cells. This may also reflect MAPK/ERK reactivation, which occurs in some cell lines, particularly after treatment with Palbociclib and Gedatolisib in combination.Of note, there was also an increase in Ribosomal S6 Kinase, in particular with the combination treatment compared to control arm, e.g.,, in DLD−1 [fold change = 1.65 ± 0.20; RPPA adjusted p-values after 4 h of treatment are shown in Supplementary Table S3. Additional RPPA analysis after 30 min of treatment was also carried out and p-values for the comparison of all antibodies measured between 30-min and 4-h treatment timepoints are shown in Supplementary Table S4.22 In this study, we investigated the impact of combining Palbociclib (CDK 4/6 inhibitor), with either Gedatolisib  or PD0325901 (selective MEK 1/2 inhibitor) on CRC cell line growth. We also investigated the proteomic effects of the combination Palbociclib with Gedatolisib in CRC cell lines with various mutational backgrounds to identify potential biomarker(s) for this novel therapy.Resistance to anti-cancer therapies evolves dynamically within 6–12 months of starting treatment due to various mechanisms, and combined drug treatment is a promising strategy for tackling resistance.50 values for single-agent Gedatolisib were between 76 and 7200 nM. These values were comparatively higher than previous studies, which reported IC50 values of less than 100 nM,24 although Caco−2, LS411N, LS1034, and SNUC4 were not included in this analysis. We also observed a higher range of IC50 values for single-agent Palbociclib in our cell lines, in comparison to other studies.25 PD0325901 demonstrated a relatively low IC50 range in our cell lines, suggesting more innate sensitivity to MEK inhibition.The ICKRAS mutated) and DLD−1 (co-occurring KRAS with PIK3CA mutated) cells. These are important findings since KRAS and PIK3CA mutations occur in approximately 60% and 20% of CRC, respectively.26 In LS411N (BRAF V600E mutated) cells, the combination Palbociclib with Gedatolisib demonstrated mild synergy whilst the combination Palbociclib with PD0325901 failed to show any synergistic effects (CI = 0.64 versus 14.7). In LS1034 cells, we noted a synergistic effect with both drug combinations, however the combination Palbociclib with Gedatolisib appears to be superior (CI = 0.11 versus 0.29). In the Caco−2 wild-type cells, both drug combinations demonstrated comparable synergistic effects. Taken together, we considered the combination of Palbociclib with Gedatolisib to be of higher priority for future clinical development, compared to the combination of Palbociclib with PD0325901 and thus decided to focus our proteomic investigations on Palbociclib with Gedatolisib. The subnanomolar IC50 range of Gedatolisib makes it a more favorable companion drug than PD0325901 to avoid excessive overlapping toxicities. Furthermore, strong efficacy and safety data is already available from Phase I clinical trials involving the combination Palbociclib with Gedatolisib.8 In these trials, Palbociclib (125 mg) was administered orally, daily for 3 weeks with Gedatolisib (110 mg) administered intravenously once during the 4-week cycle.Our results demonstrated that the combination Palbociclib with either Gedatolisib or PD0325901 exhibits synergistic anti-proliferative effects, relative to the single agents, in all cell lines including LS1034  across all cell lines. There was also a significant suppression of S6rp (S235/S236) in Caco−2, DLD−1, and LS1034 cells. This suppression was much more marked with the combination Palbociclib with Gedatolisib than with either single agent alone. Suppression of S6rp was not associated with any increased expression or phosphorylation of AKT, indicating that there was no upstream PI3K reactivation in response to the combination. We thus believe that the combination Palbociclib with Gedatolisib acts primarily at the level of mTOR, which is a downstream effector of the PI3K/AKT signaling cascade. Our results also show that the combination Palbociclib with Gedatolisib likely exhibits stronger inhibition of the PI3K/AKT/mTOR pathway in comparison to control or single-agent therapy. For example, we observed increased levels of AKT (S473) and PDK1 (S241) in some cell lines with single-agent treatment, possibly reflective of feedback loop activity.et al., 27 S6rp is considered a better surrogate biomarker of mTOR activity in comparison to p70S6K, also known as Ribosomal protein S6 kinase beta−1 (S6K1). This is because p70S6K has structural similarity to p90S6K, which is not phosphorylated by mTOR.28 As a key component of the PI3K/AKT signaling cascade, mTOR plays a crucial role in the regulation of energy metabolism and protein synthesis by directly activating p70S6K.29–32 p70S6K is a serine-threonine kinase that controls S6rp phosphorylation at five serine residues , leading to initiation of protein synthesis.33 In contrast to the S240/244 residues which are solely regulated by p70S6K, phosphorylation at the S235/S236 residues is controlled by multiple kinases including p70S6K, p90RSK, and PKA.34 This may explain the suppression of S6rp (S235/S236) which was significant in Caco−2, DLD−1, and LS1034 but not in SNUC4 cells, in contrast to S6rp (S240/S244).In view of the global suppression of S6rp (S240/S244) in all tested cell lines, this may be a promising predictive marker of clinical responsiveness for this combination therapy. As reported by Iwenofu 1 to S phases, leading to increased cell death.35–38 Interestingly, we did not observe any increase in pRb (S807/S811) following treatment with the combination Palbociclib with Gedatolisib. This is consistent with results previously described by Vora et al. 39 It appears that PI3K inhibition suppress AKT phosphorylation but can fail to suppress CDK 4/6 activity, as measured by Rb phosphorylation.40 Nonetheless, the regulation of Rb function by phosphorylation during cell cycle is not fully understood. Rb in mammalian cells has 15 known phosphorylation sites and it appears that Rb phosphorylation at specific sites is required for Rb to regulate apoptosis.42In vitro studies have shown that phosphorylation of Rb at S608/S795 in addition to S807/S811 may play a role in the induction of apoptosis.43 Furthermore, there is evidence to suggest that dephosphorylation of Rb has been widely observed during apoptosis.44 This may explain the equivocal level of pRb (S807/S811) we observed in our RPPA analysis.Emerging experimental data has suggested utilizing PI3K/mTOR inhibitors to induce non-cell autonomous actions by modulating signal transduction during GUnlike the single agents, the combination Palbociclib with Gedatolisib induced (early) pro-apoptotic effects, as demonstrated by increased BAX and Bcl−2 levels in most cell lines tested. The increase in both markers was significant in DLD−1 and SNUC4 cells but was not significant in LS1034 cells, suggesting that the magnitude of effect may be dependent on the cells’ mutational status. We did not observe any increase in caspase 3, caspase 8, cleaved caspase 7, cleaved caspase 9, or cleaved PARP levels, which are indicative of total apoptosis. However, it is possible that the 4-h timepoint used for our analysis was too early to evaluate the proteomic alterations related to the late stage of apoptosis.Finally, we observed EGFR and RSK upregulation in all cell lines after 4 h of drug treatment, which may be associated with upstream MAPK/ERK reactivation. Total EGFR and RSK upregulation were observed with both mono- and combination therapy in some of the cell lines. This suggests that the mechanism promoting resistance to PI3K-targeted inhibitors (which was Gedatolisib in this study) include feedback loops, which lead to reactivation of membrane RTKs and the contralateral MAPK/ERK pathway. This further supports our hypothesis that a multiple target inhibition strategy, rather than single agent, is a better therapeutic option to prevent the development of resistance. The combined drug inhibition using Palbociclib with Getatolisib would simultaneously target the upstream and downstream effectors including the interconnection points between the PI3K/AKT/mTOR and MAPK/ERK pathways. It provides broader inhibition on the vast interconnection of both pathways, while minimizing feedback loops activation.in vitro study and limited to five cell lines. Second, not all specific exon mutations were tested in this study, specifically PIK3CA mutations in exon 20 which may be biologically more relevant than exon 9 mutations from an epidemiology standpoint.45–47 Third, the RPPA analysis with 40 preselected antibodies was performed at only two timepoints  post-drug exposure. The RPPA results are dependent on the selected timepoints, and it is likely we will be able to capture additional proteomic information if longer timepoints are used. The specific mechanism of synergism for the combination Palbociclib with Gedatolisib could not be completely defined in this study; however, from our RPPA analysis there are several possible mechanisms, including more complete inhibition of protein synthesis-related signaling ) and increased activation of early apoptotic signaling. Despite these limitations, our study has produced evidence to support further in vivo evaluation, which is in progress.It is important to note the limitations of our study. First, it was an wild-type and mutated CRC cell lines, relative to the single agents. Our results offer good rationale for further in vivo study and clinical development of Palbociclib and Gedatolisib as emerging therapeutics in metastatic CRC patients. S6rp (S240/S244) may be a marker of responsiveness for this novel combination therapy.In summary, the novel combination Palbociclib with Gedatolisib displays clear synergistic anti-proliferative effects in both PIK3CA, KRAS, and BRAF mutations), DLD−1 (KRAS/PIK3CA mutated), LS411N (BRAF mutated), and LS1034 (KRAS mutated) were obtained from the American Tissue Type Collection . SNUC4 cells (PIK3CA mutated) were obtained from the Korean Cell Line Bank . The mutational status of the cell lines was determined using the Cancer Cell Line Encyclopedia (CCLE), and mutations were verified by us using the MassARRAY system (Agena Bioscience).Five human CRC epithelial cell lines with commonly found mutational variations were used in this study . Caco−2 KRAS G13D and PIK3CA E545K mutations. LS411N cells have a BRAF V600E mutation. LS1034 cells have a KRAS A146T mutation. SNUC4 cells have a PIK3CA E545G mutation. The cell line identity was confirmed by DNA fingerprinting (Biosciences). Cell lines were mycoplasma tested before and after in vitro experiments. Species information, source and culture details are shown for all cell lines in Supplementary Table S1.Caco−2 cells were wild type for mutations in PI3K/AKT/mTOR and MAPK/ERK pathways. DLD−1 cells have Palbociclib (CDK 4/6 inhibitor), Gedatolisib , and PD0325901 (selective MEK 1/2 inhibitor) were purchased directly from Selleckhem . All drugs were prepared in 100% dimethylsulfoxide (DMSO) at stock concentrations of 10 mM, 5 mM, and 10 mM, respectively. The two drug combinations tested were Palbociclib with Gedatolisib versus Palbociclib with PD0325901.48 Cells were plated at 1 × 104 cells/mL into 96-well plates (100 µL per well) and incubated at 37°C with 5% CO2 for 24 h. Two hundred microliters of sterile H2O was added around the edges of the plate to prevent it from drying out. Following 24-h incubation, drugs were added at the indicated concentrations and incubated at 37°C for 5 days (120 h). On Day 6, all the drug was removed, washed, and processed for absorbance measurement at 405 nm using a 96-well plate reader. Inhibition of proliferation was calculated relatively to untreated controls to obtain the dose of half maximal inhibitory concentration (IC50). A minimum number of triplicate biological assays was performed for each experiment. IC50 values were calculated using CacuSyn software. The individual inhibitor IC50 values were used for dosing guidance in the subsequent drug combination analysis. The same cell number (1×104 cells/mL) was used for the drug combination analysis. Drug concentrations used for the combination assays are shown in The acid phosphatase assay was used to test the anti-proliferative effects of Palbociclib, Gedatolisib and PD0325901, alone and in combination in each cell line. This was performed over 6 days period, as previously described.5 cells were seeded in 6-well plates and grown until confluent. The total protein was extracted using 100 µl lysis buffer , as previously described by us.50 Protein was quantified by the bicinchoninic acid (BCA) assay and stored at −80◦C until analysis. The combination Palbociclib with Gedatolisib was selected for RPPA investigation because this combination demonstrated synergism in all cell lines tested in our study.4 × 10Cell lysates with a protein concentration of 1.5 µg/µL for each replicate were prepared. Before RPPA processing, each sample was solubilized in sodium dodecyl sulfate (SDS) sample buffer  and heated to 80°C for 5 min. RPPA analysis was carried out using triplicate biological replicates following 4-h treatment with Palbociclib, Gedatolisib, and their combination in Caco−2, DLD−1, LS1034, and SNUC4 cell lines. The cell lines were treated at the same fixed ratio doses used in the drug combination proliferative analysis. Details of all primary antibodies used for RPPA analysis are detailed in Supplementary Table S2.50 and combination index at effective dose 50 (CI) values. The CI values were determined using the Chou-Talalay equation on CalcuSyn. A CI < 0.9 is considered synergistic, between 0.9 and 1.0 is additive and > 1.1 is antagonistic. Each experiment was repeated 3–4 times. For the RPPA experiments, the mean and standard error of mean (SEM) were calculated from three biologically independent protein samples analyzed on the same RPPA slide. The mean and SEM were normalized to the vehicle-treated control samples. Based on our internal precision studies, this allows us to detect changes in protein expression with a coefficient of variance (CV) of less than 20%.51 To evaluate the effect of the combination treatment, a one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test was used (GraphPad PRISM version 8). To compare the effects of Palbociclib, Gedatolisib, and their combination treatment on protein expression and phosphorylation, the Kruskal–Wallis non-parametric test was used. P values of < 0.05 were considered statistically significant.Microsoft Excel software was used to record all the raw datasets. CalcuSyn software version 3.1 (Biosoft) was used to calculate ICClick here for additional data file.


With technological advancements, radiotherapy (RT) has become an effective non-surgical treatment for hepatocellular carcinoma (HCC), comprehensively improving the local control rate of patients with HCC. However, some patients with HCC still experience radio-resistance, cancer recurrence, and distant metastasis following RT. Our previous study has revealed that hexokinase 2 (HK2), a potent oncogene, was overexpressed in radio-resistant HCC cell lines; however, its role in HCC radio-resistance remains elusive. Here, we confirmed the upregulation of HK2 in HCC tissue, which is related to unfavorable prognosis in patients with HCC, and demonstrated that HK2 exerts a radio-resistant role by attenuating apoptosis and promoting proliferation in HCC cell lines. HK2 downregulation combined with ionizing radiation showed an excellent synergistic lethal effect. Mechanistically, HK2 alleviated ionizing radiation-mediated apoptosis by complexing with pro-apoptotic protein aminoacyl tRNA synthetase complex interacting multifunctional protein 2 (AIMP2) while enhancing its autophagic lysosomal-dependent degradation, thereby increasing radio-resistance of HCC. Pharmacologically, ketoconazole, an FDA-approved antifungal drug, served as an inhibitor of HK2 and synergistically enhanced the efficacy of RT. Our results indicated that HK2 played a vital role in radio-resistance and could be a potential therapeutic target for improving RT efficacy in HCC. Hepatocellular carcinoma (HCC) is a major global health challenge and the fourth leading cause of cancer-related deaths worldwide . RadiatiResistance to IR is poly-modal and associated with biological alterations in both the innate tumor and surrounding microenvironment . IncreasHexokinase 2 (HK2), a key enzyme in glycolysis, could catalyze the following rate-limiting step in aerobic glycolysis: glucose phosphorylation and production of glucose-6-phosphate . In pancIn the present study, we observed that HK2 protein levels were elevated over time in inherent IR-R (QGY7701) HCC cell lines, whereas they were maintained in radio-sensitive HCC cell lines MHCC97H and MHCC97L. Additionally, HK2 was not upregulated over time in acquired IR-R cell lines Fig. , possiblTo assess HK2 expression in patients with HCC, we analyzed public Gene Expression Omnibus (GEO) databases (accession GSE14520 and GSE36376). Increased HK2 mRNA levels were observed in HCC tissues compared with that in adjacent normal tissues Fig. . Kaplan–To further investigate HK2-mediated radio-resistance, we constructed stable HK2-overexpression cells in radio-sensitive cell lines (MHCC97H and MHCC97L) and stable HK2-knockdown cells in radio-resistant cell lines . Western blotting verified transfection efficiency  assay of the phosphorylation of histone 2 A (pH2AX-Ser139), an indicator of effective DNA damage repair, was performed at the indicated time. The nuclear pH2AX-Ser139 focal point dissipated faster in LV-HK2 cells than in parallel control cells, whereas shHK2 in radio-resistant cells exerted the opposite effect Fig. .Because IR can lead to cell death, flow cytometry (FCM) of apoptosis was performed on cells with different levels of HK2. Although HK2 expression did not significantly affect apoptosis in the absence of IR, LV-HK2 could reduce IR-induced apoptosis Fig. . ConsistBased on in vitro results, we investigated the role of HK2 in vivo by establishing subcutaneous xenograft models in nude mice. No significant difference in proliferation was observed between subcutaneous tumors with shHK2 and those with LV-HK2. However, treatment with IR showed that HK2 overexpression could effectively enhance radio-resistance, whereas knockdown of HK2 elevated radio-sensitivity and inhibited tumor growth Fig. . We obseWe further incorporated murine-derived H22 HCC cells into subcutaneous xenograft models in nude and C57BL/6 mice. In nude mouse models, the growth rate of the HK2-overexpression group was higher than that of the parallel control group after IR Fig. . By examTo explore molecular mechanisms underlying HK2-mediated radio-resistance, immunoprecipitation (IP), and silver staining assays were carried out by comparing anti-HK2 products with anti-IgG IP products in lysates prepared from post-IR MHCC97H LV-RFP and LV-HK2 cell lines , a multifunctional tumor suppressor, can prompt apoptosis and repress cell stemness –17. WestTo confirm the relationship between HK2 and AIMP2 in unedited cells, Co-IP was performed. We observed that HK2 and AIMP2 were co-localized at the protein level only after IR Fig. . SimultaBecause AIMP2 protein levels were inversely regulated by HK2, and HK2 may mediate AIMP2 downregulation via post-transcriptional modification, we pretreated radio-sensitive LV-RFP and LV-HK2 cells with autophagy inhibitor chloroquine (CQ) or proteasome inhibitor MG-132 followed by IR. CQ treatment successfully reversed AIMP2 downregulation, whereas MG-132 treatment did not Fig. . PreviouTo further investigate the participation of autophagy in AIMP2 degradation, the expression of classic autophagy-related proteins, including LC3, P62, and Beclin1, were evaluated by western blotting. When combined with IR, LV-HK2 upregulated Beclin1 levels, enhanced LC3 II lipidation, and downregulated P62 levels compared with those in LV-RFP groups; while shHK2 had the opposite effects Fig. . IF of LWe hypothesized that the role of HK2 in radio-resistance was partly mediated by impaired AIMP2 functions. Thus, we constructed HCC cell lines with different AIMP2 and HK2 statuses. Firstly, we screened the most efficient siRNA segments and transfected them into shGFP and shHK2 cell lines  of the NF-κb pathway at the mRNA level but not at the protein level after IR  and Wnt/β-catenin signaling  pathways did not show consistent trends at mRNA or protein levels , a commonly used FDA-approved antifungal drug, inhibits glioblastoma cell proliferation by targeting HK2 . To veriSubsequently, subcutaneous xenograft models of nude mouse with MHCC97H LV-HK2 cells or H22 LV-HK2 were established. Keto was injected intraperitoneally at the beginning of the day before IR treatment and maintained every two days at a concentration of 20 mg/Kg Fig. . AlthougRT combined with systemic therapy has become the first-line treatment for patients with advanced HCC . HoweverPKM2 and LDHA, HK2 could perform functions other than regulation of glycolysis [HK2 is commonly upregulated in various cancers and exerts pro-cancer properties . We obseycolysis , 39. HK2ycolysis , 33. HK2ycolysis , 40, 41.ycolysis , 43. HK2ycolysis , 44.Our investigation revealed that HK2 levels were elevated in HCC tissues and predicted unfavorable prognosis in patients with HCC. HK2 overexpression in radio-sensitive HCC cells significantly elevated proliferation and reduced apoptosis, whereas HK2 knockdown in IR-R HCC cells induced sensitivity to IR both in vitro and in vivo. These results strongly supported the function of HK2 in radio-resistance in HCC.IR damages double-stranded DNA, leading to cell apoptosis . The proConsidering that HK2 downregulates the protein but not the mRNA level of AIMP2, we hypothesized that HK2 may mediate the degradation of the AIMP2 protein. Rescue tests using the autophagy inhibitor CQ and the proteasome inhibitor MG-132 clarified that HK2 upregulated autophagy and, thus, the degradation of AIMP2 after IR. Lysosomal autophagy, a highly conserved process of self-digestion, mediates cellular homeostasis by degrading and recycling damaged organelles and proteins in response to stress . IR can HK2 was a significant target for anti-tumor therapy. 2-deoxyglucose (2-DG), an indirect pharmacological inhibitor of HK2, has entered phase II clinical trials . Phase IThis study sheds light on the role of HK2 in HCC radio-resistance Fig. . Targetin = 54) and the latter 40% of patients were designated as the HK2-low group (n = 39). The datasets involved were downloaded from the public GEO (Gene Expression Omnibus) databases. The GSE14520 dataset contains paired tissue data from 220 patients, and tumor tissue data were sorted according to the mRNA expression. The first 50% of patients were designated as the HK2-high group (n = 110), and the second 50% were designated as the HK2-low group (n = 110).Twelve pairs of HCC and non-tumor-adjacent samples from patients undergoing curative surgery obtained from Hepatobiliary Surgery of Nanfang Hospital were used for western blot analysis. A liver cancer tissue microarray (TMA) containing 93 liver cancer samples and 87 matched normal liver tissues or cirrhosis tissues purchased from Shanghai Xinchao were used for immunohistochemistry (IHC) and prognosis analysis. IHC data of TMA were sorted from highest to lowest. The top 60% of patients were designated as the HK2-high group . Flag-tagged HK2 overexpression vectors (LV-HK2) and control vectors (LV-RFP) were transferred into MHCC97H, MHCC97L, and H22. Consistently, HK2 shRNA (shHK2) and control short hairpin RNA (shGFP) were transfected into MHCC97H IR-R, MHCC97L IR-R, and QGY7701. AIMP2 overexpression vectors (LV-AIMP2) and control vectors (LV-NC) were transferred into MHCC97H (LV-RFP&LV-HK2) and MHCC97L (LV-RFP&LV-HK2) cells. The small interfering RNA (siRNA) oligonucleotides constructed by Ribobio  contain siAIMP2 (three targets) and siControl (siNC). The plasmid of mCherry-GFP-LC3 II was constructed by Hanyi Biotech . Both were transfected into cells using Lipofectamine 3000 (Thermo Fischer Scientific) transfection reagent following the supplier’s recommendations. Ketoconazole, chloroquine (CQ) and MG-132 were all purchased from Selleck and dissolved in DMSO.The HCC cell lines were purchased from Shanghai Cell Bank. Acquired radio-resistant cell lines MHCC97H IR-R and MHCC97L IR-R were constructed as follows . These c6) were implanted subcutaneously into the backside of nude mice or C57BL/6 mice ; IR facilitation was performed on days 12 and 13 after implantation. When the volume reached 200 mm3, mice were randomly divided into four groups for further treatment. Ketoconazole (purchased from Selleck) was dissolved in 30% propylene glycol, 5% Tween 80, and 65% D5W and injected intraperitoneally at the beginning of the day before IR and maintained every two days at a concentration of 20 mg/Kg [3) was measured and calculated as follows: tumor volume = L × W × H/2, where L is length, W is width, and H is height. When the volume reached 1000 mm3, tumors were harvested and dipped into formalin immediately for further analysis. We were blinded to the group during the experiment.Stably transfected cells (5 × 10(A+B)/(EA + EB − EA × EB), where Q = 0.85–1.15 means additive, Q > 1.15 means synergism and Q < 0.85 means antagonism. E(A+B) is the cell killing rate of combination therapy, EA and EB indicate cell killing rates of A and B, respectively [-D/D0) N, where SF is the surviving fraction and D is the IR dose [The mice and cells were exposed under different irradiation doses from 0 to 8 Gy using a linear accelerator . For the CCK8 assay, cells were plated on 96-well plates 24 h before the IR and measured with cell counting kit-8 (DOJINDO Laboratories) at 450 nm for 5–6 days. Synergistic killing effects of IR and Keto were calculated using the formula: Q = Eectively , 35. Forectively . The dos IR dose .Total proteins were extracted by RIPA buffer , lysed from cells or tissues with the addition of protease inhibitors  and phosphatase inhibitors , separated by 10% SDS PAGE or 12% SDS PAGE, and then transferred onto PVDF membranes. After incubation with primary antibodies overnight at 4 °C, membranes were washed three times with PBST. These blots were incubated with HRP-conjugated secondary antibody  for 1.5 h at 4 °C. The blots were washed three times with PBST and visualized by enhanced chemiluminescence . Primary antibodies used for western blot were listed as follows: GAPDH , HK2 , AIMP2 , Cyto c , Caspase 3/p17/p19 , Bcl-xL , LC3 , P62 , Becline1 .IHC staining of tumors was performed as described previously . PrimaryTotal RNA from cells was derived using TRIzol reagent  and reverse transcribed into cDNA using Evo M-MLV RT Master Mix  according to the manufacturer’s instructions. Subsequently, mRNA expression was analyzed by using SYBR® Green Premix Pro Taq HS qPCR Kit (Rox Plus)  under a LightCycler 96 detection system (Roche). GAPDH was selected for normalization. Primer sequences were listed in Supplementary Table Cells were pretreated with the corresponding experimental design, then harvested and stained according to the manufacturer’s instructions (KeyGEN), followed by flow cytometry for apoptosis analysis , the apog for 5 min at 4 °C, and the beads complex was washed five times with PBS. Then, protein A/G magnetic beads were eluted by boiling in 5X SDS sample buffer before western blot.Total protein was extracted using cell lysis buffer with the addition of protease inhibitors  and phosphatase inhibitors . Then the lysate (2000 μg protein) was incubated overnight at 4 °C with anti-HK2 , anti-AIMP2 , or IgG . The protein-antibody complex was incubated with protein A/G magnetic beads for 6 h at 4 °C. Immunoprecipitation was then collected by centrifugation at 2000×Silver staining was performed following the protocol provided by Beyotime Technology. Briefly, the gels were fixed in 50% ethanol/10% acetic acid for 40 min after electrophoresis and washed in 30% ethanol for 10 min and Milli-Q water for another 10 min. The gels were incubated with silver staining sensitizer for 2 min and then with silver nitrate for 10 min. Afterward, the gels were put in Milli-Q water for 1 min, removed, and in the developing solution. When clear staining was achieved, gels were transferred to the stop solution for 10 min. Stained gels were stored in Milli-Q water at 4 °C .Cells were grown in confocal dishes for 48–72 h and proliferated for two to three generations, then fixed using 4% paraformaldehyde for 30 min, washed three times with PBS, and permeabilized using 0.3% Triton X-100 for 8 min. Then blocked with goat serum, followed by the appropriate antibody. Anti-HK2 antibodies , anti-AIMP2 , anti-LC3B  antibodies were used herein, followed by Alexa-488-conjugated goat anti-Mouses secondary antibody  or Alexa-555-conjugated goat anti-Rabbits secondary antibody  for imaging. The cells were then stained with DAPI as a nuclear indicator and imaged with a confocal laser scanning microscope (Olympus FV1000). Autophagy was measured by an observer blinded to experimental conditions.Cells were digested and collected after 48 h of exposure to 8 Gy irradiation, then fixed by special solution for TEM characterization. Samples were prepared on nickel or aluminum TEM grids.t-test was utilized to compare variables of two groups, and one-way or two-way ANOVA was used for multi-group comparisons. Co-localization of HK2, AIMP2, and the LC3 II punctae in IF staining were calculated with Image-Pro Plus 6.0. Significant differences were showed with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical details are included in the respective figure legends.The results were shown as mean ± SEM, and all data followed a normal distribution with homogenous variance. Survivals related to HK2 expression in patients with HCC were calculated by the Kaplan–Meier survival analysis. A two-tailed, unpaired, or paired Student’s Extended Figure1 Extended Figure2 Extended Figure3 Extended Figure4 Extended Figure5 Extended Figure6 Supplementary FigureSupplementary Table 1Supplementary Figure1Supplementary Figure2Supplementary Figure3Supplementary Figure4Supplementary Figure5Supplementary Figure6Extended Supplementary Figure1 Extended Supplementary Figure2 Extended Supplementary Figure3 Extended Supplementary Figure4 Extended Supplementary Figure5 Extended Supplementary Figure6 Original Western Blotsaj-checklist


Glioblastoma multiforme is an aggressive brain tumor with a poor survival rate despite modern therapeutic options. In this context, advanced preclinical models and the development of new treatments are urgent. Three-dimensional cultures offer new possibilities for understanding the tumor’s biology. They mimic the tumor microenvironment and its complexity, thus reflecting the patients’ neoplasm more closely. We developed a 3D model to analyze the radiation sensitivity in patient-derived glioblastoma cells.TM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedis Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. The current standard therapy consists of surgical resection, chemotherapy, tumor-treating fields (TTF), and radiation treatment ,2,3. DesThe malignancy of the tumor relies on its infiltrative nature, resistance to chemo- and radiotherapy, and intratumoral heterogeneity . A functCultivation and characterization of patient-derived GBM cells could facilitate the individualized study of this cancer type for developing novel therapies and may help to predict patients’ response to chemo- and radiation therapy . As a keIn conventional two-dimensional (2D) cell culture systems, monoclonal tumor cell lines grow in a homogeneous monoculture. This may not reflect the architecture of the patient’s tumors . AdditioIn the last few years, the development of three-dimensional (3D) cell culture models has rapidly expanded . There iIn order to establish a reliable 3D model of patient-derived GBM cells for analysis of long-term tumor growth and radiation response, we used a conical agarose 3D microwell system ,29. The 2O, 5% CO2, and 37 °C.The established GBM cell line LN229 was cultivated in Dulbecco’s Modified Eagle’s Medium with 10% FCS and 1% penicillin/streptomycin in a humidified atmosphere with 95% H2, and 95% humidity. In this study, three patient-derived GBM cell lines were included.To generate primary GBM cell lines, the patient-derived tumor tissue was cut into small pieces, transferred into a 6-well plate, and covered with medium-filled agarose cups . After Irradiation was performed with the RS225A X-ray device  at a dose rate of 1 Gy/1.07 min .TM . The colonies were counted with the GelCountTM software version 1.1.8.0 with a minimum limit of 50 cells per colony. The survival fraction (SF) was calculated by dividing the plating efficiency (PE) at the different doses by the plating efficiency at 0 Gy. The survival data were fitted to the following linear model (corresponding to the linear quadratic model with the quadratic term set to 0). The survival fraction SF is described by the radiation dose D and the linear coefficient α:The cells were seeded in 12-well plates and irradiated 24 h later with 0, 2, 4, 6, and 8 Gy. After two weeks, the cells were washed with PBS, fixed with −20 °C methanol, stained with 0.1% crystal violet, and scanned with the GelCountTM  matrices (1 × 1 or 2 × 2 mm with 880 or 200 microcavities) were put into 6-well plates and equilibrated for at least 2 h in a humidified incubator at 37 °C with RPMI medium. The cells were detached with accutase, passed through a cell strainer (40 μm), and counted. The cell number per cavity was chosen depending on the matrix size and the experiment. The equilibration medium was removed, and the cell suspension was applied to each matrix. After 20 min, the plates were placed in the incubator allowing the cells to settle in the cavities. The next day the matrices were transferred into new 6-well plates to minimize cell growth on the surface of the plate. The medium was exchanged once per week.Three-dimensional CoSeedisTM  every week. The images were then analyzed with the software ImageJ 1.48v. With an image resolution of 1200 dpi (corresponds to 1200 pixels in one inch), the scale was set at 1 inch = 25.4 mm = 25,400 μm. The measured area A was determined by a greyscale threshold. The minimum particle size was adjusted to exclude dust and artifacts. As the cells aggregated in the cavities as spheres, the volume V was calculated with the following formula:To analyze the volume of the spheroids over time, an image was taken with the GelCountTM 1 × 1 mm matrices in RPMI 1640 medium supplemented with 25 mM Hepes and 10% FCS according to the protocol for experiments with low cell densities. After 24 h, the cells were irradiated with 0, 2, 4, 6, and 8 Gy. To prevent precipitates, 25 mM Hepes was added to the medium, and the medium was changed every week. Weekly, images were taken with a high-resolution scanner (GelCountTM), and the volume was analyzed. The survival analysis was performed after 4 weeks of incubation with ImageJ software. The colonies were identified by a greyscale threshold. Colonies were counted with a binary readout. The 1 × 1 mm matrix consists of 880 cavities; therefore, the number of the developed colonies for each irradiation dose was divided by 880. The survival curves were fitted to the linear–quadratic model (LQM). The survival fraction SF is described by the radiation dose D, the linear coefficient α, and the quadratic coefficient β:A total of 10 cells/microcavity were seeded in the 3D CoSeedisTM 2 × 2 mm matrices with 1000 cells/microcavity in RPMI 1640 medium supplemented with 2.5% FCS according to the protocol for experiments with high cell densities. The medium was changed each week. The spheroids were fixed at the end of the experiments as indicated in 4% paraformaldehyde for 6 h and then transferred into 70% ethanol. The paraffin-embedding, slicing, and IHC staining for γH2AX (phospho-histone H2A.X antibody (Ser139), 9718S, Cell Signaling Technology , dilution: 1:500), Ki67 , dilution: 1:50) and GFAP , dilution 1:400) was performed by the Comparative Experimental Pathology, Institute of Pathology  using a Bond Rxm  with a Polymer Refine Detection Kit. All slides were digitalized using a slide scanner system . The γH2AX-stained spheroids were analyzed using the software QuPath (open source) [The cells were seeded into the 3D CoSeedis source) . The soft-test and two-way ANOVA  were applied. A p-value of ≤ 0.05 was considered to be statistically significant.The software GraphPad Prism  was used for the statistical analysis. For the comparison of the cell lines, a To analyze the radiosensitivity of patient-derived primary GBM cells, a clonogenic assay—the gold standard for the determination of radiosensitivity—was performed. As the primary cell lines showed a heterogeneous and spreading growth pattern and hence formed no compact but rather widespread patchy and overlapping colonies a, countiThree-dimensional assays constitute an attractive alternative to 2D assays as they reflect better the in vivo situation. The 3D CoSeedis™ assay (abc biopply) was selected as it offers a novel 3D approach to the traditional CFA. It consists of a conical agarose-based microwell array that allows the growth of up to 880 spheroids in one matrix.8 μm3 after 60 days. In comparison to the primary cells, the established cell line LN229 only reached a volume of 2.1 × 107 μm3 after 60 days.To monitor the 3D growth of the primary GBM cell lines  in comparison to the established GBM cell line LN229, images were taken once a week, and the volume of ~200 spheroids was measured. The time-dependent development of the volume is displayed in To illustrate the heterogeneity of the cell aggregates, the size distribution of the individual spheroids is depicted in For a morphological overview, the spheroids were grown in 2 × 2 mm 3D CoSeedis™ matrices, fixed, and stained with H&E . The estTM matrix containing 880 microcavities was chosen as it enables the analysis of up to 880 spheroids for each irradiation dose. To simulate the conditions of the 2D CFA, only 10 cells per microcavity were seeded, and 24 h later, the cells were irradiated with 0, 2, 4, 6, and 8 Gy.To investigate the radiosensitivity of primary GBM cell lines in the 3D CFA, the 1 × 1 mm 3D CoSeedisThe volumetric growth of the primary cell lines in the 3D CFA experiment was measured weekly for each irradiation dose. As expected, a dose-dependent decrease in the volume with higher irradiation doses was observed in all primary GBM cell lines . The priTo determine the survival fraction in the 3D CFA analogous to the 2D CFA, the ability to form 3D colonies in the matrices was evaluated 4 weeks after irradiation by a binary read-out. The number of microcavities containing a spheroid was divided by the number of evaluated microcavities for each irradiation dose. The respective survival data were fitted according to the LQM. In contrast to the 2D CFA, a fit of the 3D survival data with the LQM was feasible, showed positive α- and β-values and hence the survival curve revealed the expected shoulder a. The raTo analyze DNA damage and repair after irradiation, the primary GBM cell lines were seeded into 2 × 2 mm 3D CoSeedis™ matrices, grown for 5 weeks, and irradiated with 0, 4, and 8 Gy. Spheroids were fixed 1 h and 24 h after irradiation, embedded, cut and stained against γH2AX, a marker for DNA double-strand breaks (DSBs). Representative images of the γH2AX-stained spheroids 1 h after irradiation can be seen in 50 and D10 values in H74 in comparison to H75 and H77 (The percentage of γH2AX-positive cells in the spheroids 1 h and 24 h after irradiation has been quantified and depicted in  and H77 .Glioblastomas are heterogeneous and infiltrative tumors that have the ability to adapt and build resistance against existing treatments. Although novel therapeutic approaches are available for other cancer types, treatment options for this complex tumor are lagging significantly behind. Thus, preclinical models that recapitulate GBM pathophysiology and predict later clinical efficiency are one of the key challenges to accelerate progress to successful personalized treatment ,34. In cIn recent years new strategies for the development of 3D models have been implemented ,36 as thTM agarose-based system was used to culture patient-derived glioblastomas in 3D culture. This system has the advantage of being non-adhesive, so the cells can self-assemble and form a 3D culture [In this study, the 3D CoSeedis culture .TM 3D cultures can reflect the physiological growth patterns of glioblastoma tumors. This culture method offers a great opportunity for patient-derived primary cancer cells. Not only can the spheroid volume analysis of the 3D CoSeedisTM cultures present a higher resemblance to the original tumor, but it also can simplify the subsequent evaluation of multiple histological sections of the spheroids.To establish the 3D model with patient-derived GBM and to analyze the best growth conditions, the primary cells, as well as the established cell line LN229 were cultured for 60 days, and the volume of the spheroids was determined a. The diInitially, the aim of this study was to compare the radiation sensitivity measured in the 3D assay with the conventional 2D CFA in primary GBM. However, the 2D CFA evaluation was difficult due to the heterogeneous and spreading growth pattern and overlapping colonies of the primary cell lines. Furthermore, the 2D CFAs could not be fitted to the linear–quadratic model due to negative beta terms. In contrast to the 2D CFA, the survival data derived from the 3D assay could be fitted with the LQM and revealed positive beta terms.TM 3D culture offers a simple method for the evaluation of radiosensitivity with high reproducibility. A volumetric analysis of the spheroids over time showed a dose-dependent decrease in the volume, which could also be a marker for radiation response. The primary GBM cell line, H74, exhibited a smaller volume in all radiation doses compared to H75 and H77, indicating that the primary cells show patient-specific differences in the volume of the spheroids.In comparison to the 2D assay, the 3D assay has the advantage of being evaluated longitudinally at different time points without fixation. Additionally, the spheroids could also be used for histological analysis. The CoseedisAlthough the evaluation of the 2D CFA was difficult, both 2D and 3D assays demonstrated a similar radiation response for the three primary GBM cell lines. Gomez-Roman et al.  also disTM 3D culture model used in our study, the hydrogel constitutes an extracellular matrix and enables the interaction between cells and the surrounding matrix. This cell–matrix interaction might explain the enhanced radioresistance in this 3D model.Jiguet Jiglaire et al.  culturedSimilar discoveries have been made for other cancer types. The study of Koch et al.  demonstrγH2AX IHC staining of the patient-derived GBM spheroids 1 h after irradiation revealed an increase in DNA double-strand breaks after 4 Gy and a further increase after 8 Gy a. After Overall, the 3D culture system used can offer more natural conditions and a cellular environment to study cancer biology in patient-derived glioblastoma. In the future, it might provide a valuable tool to predict patients’ responses to radiation and other therapies.This study reports the establishment of a 3D culture model to analyze the radioresponse and characterize patient-derived GBM cells. The agarose-based 3D model provides a good alternative method to analyze the radioresponse of primary GBM cell lines. Especially for GBM cell lines that are difficult to analyze in the standard 2D CFA, the 3D model offers a more reliable method. Moreover, the 3D assay better recapitulates the 3D structure of a tumor, and it also provides the possibility of additional evaluations, such as measurement of the volume and histological analysis to show the 3D architecture of the spheroids. Furthermore, subsequent immunohistological staining of the spheroids can visualize, for example, the distribution of irradiation-induced DNA double-strand breaks.In summary, this 3D model may better reflect the heterogeneity and radiation response of primary GBM cells than currently available in vitro 2D models and may have a benefit for therapy prediction in personalized medicine.