Graphical abstract graphic file with name fx1.jpg [93]Open in a new tab Highlights * • Clinical radiosensitivity can be modeled with a rectal cancer organoid assay * • A drug-radiation screen identifies MEK inhibitors as potent irradiation enhancers * • MEK inhibitors downregulate the DNA damage response protein RAD51 * • Combined MEK-PARP-radiation shows efficacy in organoid and xenograft models __________________________________________________________________ Xiao et al. use patient-derived rectal cancer organoids to identify that MEK inhibitors enhance radiation sensitivity by downregulating the DNA damage response protein RAD51. They find synergy with combined PARP inhibition in different preclinical models, thereby offering a promising regimen and potential future alternative to conventional chemoradiation in locally advanced rectal cancer. Introduction Colorectal cancer (CRC) stands as a leading cause of cancer-related mortality.[94]^1 Over one-third of CRC originates in the rectum, often presenting at a locally advanced stage, which is defined as Union for International Cancer Control (UICC) classification T3/T4 (invasion beyond muscular layers) and/or node-positive disease. The current standard of care for most locally advanced rectal cancers is neoadjuvant chemoradiotherapy, followed by surgical resection of the tumor.[95]^2 Introduction of neoadjuvant chemoradiotherapy led to improved local tumor control rates in clinical trials.[96]^3 The most commonly applied regimens include a combination of long-course radiation with intravenous or oral fluoropyrimidine and, more recently, the addition of consolidation chemotherapy with 5-fluorouracil and oxaliplatin, termed total neoadjuvant therapy (TNT).[97]^4 The response to neoadjuvant chemoradiotherapy varies significantly between individuals, ranging from complete responses without detectable tumor residues to non-response. Patients who achieve complete response have an improved overall survival and may avoid surgical resection of the rectum, which can be severely debilitating. While standard neoadjuvant chemoradiotherapy has resulted in complete response rates of ∼15%,[98]^5 intensified regimens such as TNT can significantly increase the rates of complete response. However, this success is achieved at the expense of increased toxicity, which is caused by the broad mode of action of conventional chemotherapeutic agents, notably neurotoxicity induced by oxaliplatin.[99]^6 Furthermore, resistance to radiotherapy in rectal cancer has been linked to several molecular mechanisms, including enhanced DNA damage repair, apoptosis escape, regulation of cancer stemness pathways, metabolic reprogramming, and others.[100]^7 Approaches that target such mechanisms or tumor-specific alterations to enhance radiosensitivity have not been introduced into clinical practice, despite promising preclinical results with different small-molecule drugs and antibodies.[101]^8 One of the underlying reasons is the absence of suitable tumor models that adequately reflect the biological characteristics of rectal cancers, which are dominated by particularly high frequencies of RAS-MAPK (rat sarcoma virus/mitogen-activated protein kinases) and WNT pathway mutations.[102]^9 To this end, traditional 2D cell culture models often fail to fully capture the complexity of human rectal cancer biology and therapeutic responses. Recently, patient-derived organoids have been introduced as models that can recapitulate the tumor biology of many cancer types and their response to different therapeutic modalities.[103]^10 In particular, studies have shown associations of rectal cancer organoids’ response to radiation with response of corresponding tumors in patients.[104]^11^,[105]^12^,[106]^13 So far, however, rectal cancer organoid platforms have not been exploited to systematically screen for drug candidates that can enhance the response of rectal cancers to radiotherapy. In this study, we establish a rectal cancer organoid platform that recapitulates clinical radiosensitivity and use it to perform large-scale drug screens to identify drugs synergizing with radiation therapy. We observed that inhibitors of the RAS-MAPK pathway, in particular mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors, can strongly increase the sensitivity of rectal cancer organoids and CRC cell lines to radiation. Mechanistically, we find that radiotherapy induces an activation of RAS-MAPK signaling, which could be suppressed by MEK inhibition. Moreover, MEK inhibitors (MEKi) downregulate RAD51 recombinase in protein levels, a key component of the DNA repair machinery. Accordingly, we find that MEKi synergize with poly ADP-ribose polymerase 1/2 (PARP1/2) inhibitors in reducing tumor cell viability, and the combination of these two agents can further enhance the effectiveness of radiotherapy in CRC cell lines, organoids, and murine xenograft models. Results An organoid platform recapitulates essential aspects of rectal cancer To model cancer biology and identify treatment options for rectal cancer, we established an organoid-based platform and living biobank. Organoids were generated from pre-treatment endoscopic biopsies from patients with rectal cancers of different UICC/tumor-node-metastasis stages ([107]Figure 1A; [108]Table S1). They showed heterogeneous morphologies and molecular alterations that are characteristic for CRC ([109]Figure 1B).[110]^14 Previous studies have demonstrated associations between clinical signs of radiation response in patients with rectal cancer and radiation response of corresponding rectal cancer organoids, using various protocols.[111]^11^,[112]^12^,[113]^13 We established a standardized, robot-assisted radiation protocol for organoids, based on our previously published high-throughput screening platform.[114]^15 By exposing tumor organoid cultures to different doses of radiation, we observed a clear dose dependency of organoid viability and a high degree of variation in radiosensitivity between different patient donors ([115]Figures 1C–1F, [116]S1A, and S1B). The variation in response was not explained by frequent mutations found in the organoids, consistent with previous findings ([117]Figure S1C).[118]^9 Clinical response to chemoradiotherapy determined by magnetic resonance imaging (MRI) regression grading showed a significant association (almost perfect except for one outlier) with the radiation response of corresponding patient-derived rectal cancer organoids ([119]Figures 1D–1F). Additionally, histopathological analysis of regression grade in post-radiation tumor resection specimens, as well as analysis of post-treatment changes in tumor length in MRI, also showed a strong association (although not statistically significant) with organoid response ([120]Figures 1D–1F and [121]S1D). Finally, endoscopic assessment of therapy response corresponded to the radiosensitivity of organoids in our assay (representative clinical images shown in [122]Figure 1F). These findings are in concordance with observations of previous studies and support the clinical and functional relevance of our organoid radiation assay in modeling the radiosensitivity of rectal cancers. Figure 1. [123]Figure 1 [124]Open in a new tab An organoid platform recapitulates essential aspects of rectal cancer (A) Schematic illustration of the rectal cancer organoid platform and approach for association of patient and organoid response. (B) Driver mutations identified in patient-derived organoids. (C) Response of rectal cancer organoids to increasing doses of radiation. (D) Analysis of organoid response to radiation according to donor patients’ rectal cancer response to radiation therapy assessed by MRI-based regression grading (left, 11 evaluable cases, two-tailed Student’s t test) and histopathological examination (right, 12 evaluable cases, two-tailed Welch’s t test). (C and D) Each data point represents the mean of 3–6 biological replicates tested per organoid line. (E) Representative bright-field images of rectal cancer organoid cultures undergoing radiation. Scale bars: 100 μM. (F) Representative endoscopy (left) and MRI (right) images from selected patients pre-treatment and post radiation therapy, sorted according to organoid response to radiation therapy. Green dotted lines (left) and red arrows (right) indicate location of rectal cancer. See also [125]Table S1; [126]Figure S1. High-throughput screening in rectal cancer organoids identifies RAS-MAPK signaling inhibitors as enhancers of radiation response Radiation is usually combined with fluoropyrimidines as chemotherapeutic agents for the treatment of locally advanced rectal cancer. While fluoropyrimidines have been reported to exhibit radiosensitizing properties,[127]^16 their modes of action are broad and non-selective. Hence, drugs with stronger radiosensitizing effects are needed to achieve deeper responses, which may spare patients from debilitating rectal resection. To screen for synergistic treatment combinations of radiation with medical therapies, we used our rectal cancer organoid biobank and further developed our semi-automated radiation workflow toward a high-throughput assay for combined drug and radiation therapy screening. Within this workflow, organoids underwent radiation and drug treatments in 384-well format in a viability assay over 9 days ([128]Figure 2A). We used ΔAUCs (area under the dose-response curve of non-irradiated-irradiated conditions) as a simple metric to screen for drugs that could enhance the radiation effect. Normalizing perturbations to the plate-specific dimethyl sulfoxide (DMSO) controls (i.e., separately for irradiated or non-irradiated plates) revealed radio-enhancing effects and avoided overestimation of effects and bias between the screened plates. We first used two organoid lines that were resistant to radiation therapy (D080T and D007T, compare [129]Figure 1) and applied radiation treatment in combination with a library of 224 drugs, mostly kinase inhibitors, in four concentrations ([130]Figures 2B and [131]S2A–S2C). In both organoid lines, we identified several kinase inhibitors that enhanced radiation effects and also compounds that diminished tumor cell killing upon radiation treatment ([132]Figures 2C–2F). The enhancing effects were generally stronger in organoid line D080T than in D007T ([133]Figures 2C and 2E). Many compounds that conferred high radiation-induced killing in both organoid lines belonged to the class of RAS-MAPK signaling inhibitors (particularly inhibitors of epidermal growth factor receptor [EGFR] and MEK, [134]Figures 2D and 2F). To validate these findings and to identify additional clinically available radiation-enhancing drugs with the potential for fast clinical translation, we performed further screening experiments. We used a library containing 140 cancer drugs, mostly Food and Drug Administration (FDA) approved, that could be meaningfully modeled in organoid experiments (i.e., including drugs with a mechanism directly targeting cancer cells, [135]Figures 2G and [136]S2D–S2H). This library included every drug in five concentrations and was tested in 10 different rectal cancer organoid models, including both RAS-mutated and wild-type cases ([137]Figures 2H–2L). Irradiation was done with 4 Gy for most lines and 2 Gy for lines with higher radiation sensitivity. Most drugs showed similar responses in irradiated and non-irradiated conditions, while mainly inhibitors of the RAS-MAPK signaling pathway recurrently demonstrated enhanced killing together with radiation ([138]Figure 2H). Ranking drugs according to ΔAUC demonstrated that RAS-MAPK (EGFR and MEK) pathway inhibitors, as well as PARP inhibitors (PARPi), which are known radiosensitizers in different tumors,[139]^17 were among the strongest hits (ΔAUC ± SEM trametinib = 0.0934 ± 0.0286, afatinib = 0.1488 ± 0.0388, talazoparib = 0.1064 ± 0.0211, [140]Figures 2I–2K, [141]S3A, and S3B). The degree of radiosensitization varied between the different organoid models, likely representing the molecular heterogeneity of rectal cancers ([142]Figures 2L and [143]S3B–S3D). With respect to MEKi, seven out of ten organoid lines showed enhanced tumor organoid killing to varying degrees when combined with radiation ([144]Figure 2L). In six of them, we observed a significantly increased ΔAUC of MEKi trametinib in irradiated vs. non-irradiated condition compared to the average ΔAUCs of all tested drugs ([145]Figure S3D). EGFR inhibitors exhibited similar trends but not congruent profiles of radio-enhancement across different organoid lines (3 of the lines showed significantly increased ΔAUCs for afatinib, [146]Figure S3D), while the profiles were distinct for PARPi (5 of 10 lines had significantly enhanced ΔAUCs upon treatment with talazoparib; [147]Figures 2L and [148]S3D). We also tested associations of common molecular alterations in organoid lines with the level of radiosensitization. We found that organoid lines with wild-type TP53 status generally showed drug responses that were more strongly modifiable by radiation, especially with MEKi, while no association of radiation enhancement was observed with RAS mutation status ([149]Figures S4A and S4B). Figure 2. [150]Figure 2 [151]Open in a new tab Drug screening identifies RAS-MAPK pathway inhibitors to synergistically enhance radiation in rectal cancer organoids (A) Schematic representation of the drug-radiation screening workflow. Rectal cancer organoids were seeded in 384-well plates; drug perturbations with 4–5 concentrations and radiation (2–4 Gy) were performed on day 3, before viability was measured on day 9 after seeding. Interactions of drugs and radiation were analyzed by calculating the difference between areas under the dose-response curves (ΔAUC values) between irradiated and non-irradiated conditions, each normalized to respective irradiated and non-irradiated DMSO controls on the same plates. (B) Composition of the kinase drug library with 224 drugs, tested in 4 concentrations in two organoid lines. (C) Ranking of differential effects of kinase inhibitors with or without radiation in tumor organoid D080T. (D) Top 10 hits with highest radiation enhancement (ΔAUC values) in the kinase library screen of organoid line D080T. (E) Ranking of differential effects of kinase inhibitors with or without radiation in cancer organoid D007T. (F) Top 10 hits with strongest radiation enhancement (ΔAUC values) in the kinase library screen of organoid line D007T. (C–F) Mean values of two biological replicates are shown. (G) Composition of the clinical cancer library consisting of 140 drugs. The drugs were administered in 5 concentrations, and 10 organoid lines were tested. (H) Mean area under the curve of all drugs tested in the clinical library in radiated vs. non-radiated conditions. (I) Ranking the mean differential effects of clinical cancer drugs with or without radiation in 10 rectal cancer organoids. (J) Top 10 hits with strongest radiation enhancement (mean ΔAUC values) in the clinical library screen with ten rectal cancer organoids. (K) Distribution of ΔAUCs of selected groups of inhibitors. (L) ΔAUCs of individual organoid lines, ΔAUCs of MEKi and PARPi are highlighted. (I–K) Mean values of 10 tested organoid lines are shown. (H and L) Each data point represents the mean ΔAUC value of two biological replicates tested for each organoid line. See also [152]Figures S2–S7. In conclusion, high-throughput drug-radiation combination screens with both kinase and clinical libraries independently showed a strong enhancement of radiation with inhibitors of RAS-MAPK signaling, especially MEKi, in the majority of tested organoid lines. MEK inhibition is synergistic with radiation in CRC cell lines and organoids Among the identified drug candidates, MEKi showed the strongest enhancement by radiation. MEKi targets RAS-MAPK signaling downstream of oncogenic RAS mutations, which are highly prevalent in rectal cancers.[153]^9 In two previously tested organoid lines (D080T and D007T), radiation with 4 Gy combined with the FDA-approved MEKi trametinib resulted in a decrease of cell viability, which was significantly stronger in combination with radiation treatment ([154]Figures S5A and S5B). To prove a synergistic effect of MEKi with radiation, we calculated the expected combination response of both perturbations for each tested concentration using a Bliss independence model, as recently reported for drug-drug combinations.[155]^18 This revealed a clear excess of the experimentally observed combination response over the expected response, proving synergy between radiation and MEKi. Microscopy images of organoids showed corresponding phenotypes, with a reduction of organoid size and number after combination therapy ([156]Figures S5A and S5B). Replicative cell death is a major cause for the antineoplastic effect of radiotherapy. To test radiation effects over several cycles of cell proliferation, we performed complementary experiments in CRC cell lines using viability assays and gold-standard colony forming assays (S5C-E, S6A). We selected three commonly used CRC cell lines SW480, DLD1, and HCT116 with different genetic backgrounds and degrees of intrinsic radiosensitivity ([157]Figure S6A). Selection was also based on the presence of Kirsten rat sarcoma virus (KRAS) mutations in all three cell lines, as KRAS mutations are highly prevalent in rectal cancers and we assumed that MEK1/2 inhibition would be more potent in models with activated RAS-MAPK signaling.[158]^9 Radiation was performed using sublethal doses of 2–4 Gy, depending on the intrinsic radiosensitivity of the cell lines ([159]Figure S6A). Measurement of cell viability 5–6 days post radiation and in the presence of different concentrations of trametinib showed enhanced antineoplastic effects with combination therapy in all three cell lines (S5C-D). The level of radio-enhancement differed between the lines but was uniformly observed at low nanomolar concentrations of trametinib. Again, comparing the observed combination response with the expected response according to the Bliss independence model revealed a clear excess over the Bliss model in all cell lines ([160]Figure S5D). The enhancing effect of combined MEKi and radiation was confirmed in long-term colony-forming assays (10–12 days of treatment) ([161]Figure S5E). We also observed sensitizing effects for three pharmacological inhibitors of the RAS-MAPK pathway, targeting EGFR, KRAS:SOS1 (Son of Sevenless 1), and extracellular signal-regulated kinase 1/2 (ERK1/2) ([162]Figures S6B–S6F). Compared to MEKi, the radio-enhancing effect of the three compounds was weaker and more cell line dependent. When compared to the murine double minute 2 inhibitor and previously reported radiosensitizer nutlin-3a,[163]^19 MEKi could achieve similar sensitizing effects but at much lower drug concentrations ([164]Figures S7A and S7B) in TP53 wild-type HCT116 cells. Of note, nutlin-3a also showed radiosensitizing effects in our organoid assays, as compared to MEKi ([165]Figure S7C). These results indicate that targeting aberrant RAS-MAPK signaling, especially by MEKi, significantly increases cellular response to radiation in CRC. Radiation induces RAS-MAPK signaling in CRC cell lines and organoids To determine mechanisms underlying the radiosensitizing effects of MEKi, we first assessed the activity of RAS-MAPK signaling after irradiation by measuring pERK levels, as the pathway has previously been associated with radiation response in different tumor models.[166]^20 We observed that radiotherapy induced a transient increase in ERK phosphorylation in DLD1 and SW480 cell lines but not in HCT116 ([167]Figures 3A and [168]S8A). The exact onset of RAS-MAPK activation differed between cell lines and was observed most consistently between day 3 and 6 post radiation ([169]Figure 3A). Activation of RAS-MAPK signaling was also demonstrated at the level of target genes, as irradiation increased the expression of sprouty RTK signaling antagonist 2 or dual specificity phosphatase 4 in CRC cell lines ([170]Figure 3B) and organoids ([171]Figure S8B). Expression profiling of three patient-derived CRC organoid lines after irradiation with 4 Gy showed a number of differentially expressed genes, some of them related to the RAS-MAPK signaling pathway ([172]Figures 3C and [173]S8C). Using pathway enrichment analysis with Molecular Signatures Database HALLMARK gene sets,[174]^21 we found that, for instance, “KRAS SIGNALING UP” was among the significantly upregulated gene sets in irradiated organoids, in addition to signatures such as apoptosis, P53_Pathway, and several inflammatory pathways ([175]Figures 3D and [176]S8D). We found that concomitant treatment with trametinib potently repressed basal and radiation-induced increase in pERK levels and expression of target genes of RAS-MAPK signaling in CRC cell lines ([177]Figures 3E and 3F). This finding was corroborated in two CRC organoid lines, as MEK inhibition markedly suppressed ERK phosphorylation induced by radiation ([178]Figure 3G). In summary, our results suggest that activation of RAS-MAPK signaling presents a mechanism of cellular adaptation of CRC to radiation. Targeting the pathway with MEKi could abolish this adaptive activation, thus providing a mechanism by which the drug sensitizes CRC cells to radiation. Figure 3. [179]Figure 3 [180]Open in a new tab Radiation induces activation of RAS-MAPK signaling (A) Phosphorylation of ERK1/2 in CRC lines at different time points after irradiation. (B) Expression of RAS-MAPK pathway target genes is induced in DLD1 and SW480 cell lines 6 days after irradiation. (C) RNA expression profiling of rectal cancer organoid line D007T, 96 h after irradiation treatment with 4 Gy. Volcano plot of differentially expressed genes in irradiated vs. non-irradiated organoids. Target genes of the EGFR signaling pathway according to PROGENY[181]^22 are highlighted. RNA expression profiling experiments of D080T and D160T can be found in [182]Figure S7C. (D) Gene set enrichment analysis of HALLMARK[183]^21 gene sets in irradiated vs. non-irradiated organoids D007. Analysis of D080T and D160T can be found in [184]Figure S7D. (E) Phosphorylation of ERK1/2 in CRC lines after irradiation is reduced by MEKi trametinib (TRA) treatment. (F) Transcriptional induction of target genes of the RAS-MAPK pathway after irradiation is suppressed by MEK inhibition in CRC cell lines. (G) Phosphorylation of ERK1/2 in rectal cancer organoids 2 days after irradiation is reduced by concomitant MEKi treatment. (A, E, G) Representative images of three independent biological replicates are shown. (C and D) Data from five independent biological replicates are shown. (B and F) Data from three independent experiments are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 two-tailed Student’s t test. See also [185]Figure S8. MEK inhibition interferes with DNA damage response via repression of RAD51 The DNA damage response pathway is activated upon radiation-induced DNA double-strand breaks (DSBs). We assessed if targeting MEK1/2 affects this process by first measuring the formation of DSBs upon radiation and the kinetics of their resolution in the presence of the inhibitor. As shown in [186]Figures 4A and 4B, radiation rapidly caused the formation of gamma histone 2AX (γH2AX)-positive foci in the nucleus of CRC cells, which decreased over time. Concomitant treatment with MEKi directly after irradiation neither caused an increase in the number of foci per nuclei nor changed the speed of their resolution ([187]Figure 4B). This observation was confirmed by immunoblot analysis of p-γH2AX levels, which are increased upon irradiation but not reduced by MEKi ([188]Figure 4C). Next, we analyzed if subsequent steps of the DNA repair pathway are affected by MEKi. DSBs can be repaired by two distinct pathways of the DNA repair machinery,[189]^23 and we first measured transcript levels of main components of both pathways. We found that radiation upregulated the transcript levels of many DNA repair genes such as DNA damage-binding protein 2 and X-ray repair cross complementing (XRCC) 2 in CRC cell lines ([190]Figures S9A and S9B), consistent with observations from previous studies.[191]^24 We then performed global proteomics profiling of three CRC cancer cell lines. A small set of proteins were strongly downregulated by MEKi, including RAD51, a central component of the homologous recombination DNA repair pathway, which was reduced in all three CRC lines. In DLD1, we observed a marked decrease, while in the other two cell lines, the protein was nearly absent after trametinib treatment ([192]Figures 4D, [193]S9C, and S9E).[194]^25 Interestingly, protein levels of other components crucial for the repair of DSBs remained mostly unchanged, including breast cancer 1 (BRCA1), PARP2, or ataxia telangiectasia mutated ([195]Figures 4D; [196]S9E). Loss of RAD51 upon MEKi was further confirmed in all CRC cell lines and three cancer organoids lines by immunoblot, showing a dose-dependent decrease of RAD51, both in the presence and absence of radiation ([197]Figures 4E, 4F, [198]S10A, and S10B). MEKi-induced reduction of RAD51 began approximately 12 h after addition of trametinib ([199]Figure S10C) and was not caused by a transcriptional repression ([200]Figure 4G). Of note, treatment of CRC cell lines with other inhibitors of the RAS-MAPK pathway at different levels (EGFR inhibition, ERK1/2 inhibition) did not lead to changes in RAD51 levels, suggesting that the mechanism is specific to MEKi ([201]Figure S10D). Co-treatment with two proteasomal inhibitors did not rescue MEKi-induced loss of RAD51, indicating that MEKi elicits proteasome-independent mechanisms to reduce RAD51 levels ([202]Figure S10E). This result was corroborated by cycloheximide chase assays, which did not show a significant acceleration of RAD51 loss upon blockage of de novo protein synthesis ([203]Figure S10F). We also observed that radiation itself increased RAD51 protein levels within 24 h, and in some lines, such as DLD1, also at later time points (day 6) ([204]Figures 4E and [205]S10G). Hence, we hypothesized that functional depletion of RAD51 would increase radiosensitivity in our CRC models. To this end, we used RNAi to efficiently knock down RAD51 in CRC cell lines, which resulted in a clear radio-enhancement in colony forming assays ([206]Figure 4H). Moreover, we used the RAD51 inhibitor RI-1 to pharmacologically target the protein function in CRC cell lines and organoids. Similar to the RNAi-mediated knockdown, RI-1 sensitized both tumor models to irradiation ([207]Figures 4I and 4J). In summary, these results indicate that MEKi-induced loss of RAD51 is a central mechanism explaining its effect as a radiosensitizer. Figure 4. [208]Figure 4 [209]Open in a new tab MEKi modulates DNA damage response by downregulating DNA repair protein RAD51 (A) Radiation-induced DNA damage as determined by immunofluorescence staining of p-γH2AX. Green, p-γH2AX; blue, DAPI; 63× magnification; scale bars: 20 μm. (B) Measurement of p-γH2AX foci per nuclei under different treatment conditions. (C) Immunoblot showing induction of cellular p-γH2AX levels upon radiation. (D) Global proteome profiling by mass spectrometry of SW480 cells after treatment with 100 nM trametinib vs. DMSO for 24 h, abundance of selected DNA damage response pathway proteins is depicted below. (E) RAD51 protein expression at different time points after irradiation and MEKi trametinib treatment in CRC cell lines. (F) RAD51 protein expression 2 days after irradiation and MEKi trametinib treatment in patient-derived rectal cancer organoids. (G) RNA expression levels of RAD51 in CRC cell lines 24 h after irradiation ± trametinib treatment as determined by qPCR. (H) Colony-forming assay with CRC cell lines after siRNA-mediated knockdown of RAD51 ± radiation. Staining of cell culture plates was performed 11 days post radiation. Knockdown efficiency of RAD51 after 48 h is shown by western blot (left). (I) Colony-forming assay in CRC cell lines after treatment with different concentrations of the RAD51 inhibitor RI-1 for 11 days ± radiation. Scans of complete wells of standard 6-well plates are shown (9.6 cm^2 per well) (H and I). (J) Proliferation of patient-derived rectal cancer organoids after treatment with RI-1 for 2 days and ± radiation, scale bars: 50 μm. (A, E, F) representative images of three independent biological replicates are shown. (B and G) Data from three independent experiments are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, two-tailed t test, p values are only shown in case of significant differences. See also [210]Figures S9 and [211]S10. MEK and PARP inhibition have synergistic effects on viability in CRC models Besides MEKi, we also identified inhibitors of additional pathways that strongly enhanced sensitivity to radiation in our primary screen in rectal cancer organoids. We hypothesized that a combination of MEKi with one of these inhibitors could potentiate the antineoplastic effects, particularly when combined with radiation. Specifically, combinations of MEKi with EGFR and phosphatidylinositol 3-kinase (PI3K) inhibitors were previously shown to elicit synergistic antineoplastic effects.[212]^26 Therefore, we performed drug combination experiments in seven rectal cancer organoid lines (five of them previously tested, two new lines as unbiased set, all of them RAS mutated, four TP53 wild type, and three TP53 mutated) using high-resolution drug concentration matrices. To this end, MEKi was combined with four drugs representing pathways with strong positive interaction with radiation (PI3K, PARP, EGFR, and CHK1), under irradiated and non-irradiated conditions ([213]Figures 5A and [214]S11A–S11G). Focusing on drug synergy in the absence of radiation first, we determined most relevant combinations, again using the Bliss independence synergy model, and additionally tested further commonly used synergy models (highest single agent, Loewe synergy model, and zero interaction potency [ZIP] model). We found that the combination of MEKi with the PARPi talazoparib and PI3K inhibitor taselisib was consistently synergistic across the tested organoid lines, while EGFR inhibitor dacomitinib and CHK1 inhibitor MK-8776 showed less consistent effects ([215]Figure 5B). Particularly high synergy scores were noted for organoid line D080T. Thus, our assay confirmed previously observed synergies of MEK and PI3K inhibition, as well as EGFR inhibition in CRC models.[216]^26 Since we had previously shown that MEK inhibition interferes with DNA damage response via RAD51, we further focused on PARP inhibition as a combination partner that converged on the DNA repair pathway. We performed in-depth evaluation of combinations of MEKi and PARPi at different concentrations for drug synergism. Using the Bliss synergy model, we found that the drug combination was most synergistic in lower-to-medium concentrations of both PARPi and MEKi, particularly in the range of 0.039–2.5 μM talazoparib and 2.4 nM–0.16 μM trametinib in our organoid assays ([217]Figures 5C–5F and [218]S12). In this concentration range, the increased efficacy of the combination was clearly visible by comparing the observed response to the expected response according to the Bliss synergy model ([219]Figure 5G). We also confirmed these findings using two CRC cell lines, in both short- and long-term viability assays ([220]Figures 5H and 5I). Synergistic antineoplastic effects in short-term proliferation assays were observed in both Bliss and ZIP synergy models (maximum Bliss score: DLD1 18.68, SW480 38.41; maximum ZIP score: DLD1 15.57, SW480 44.47). Together, these results indicate that MEKi and PARPi can synergistically reduce viability in different CRC models at low concentrations, even in the absence of radiation. Figure 5. [221]Figure 5 [222]Open in a new tab MEK and PARP inhibition have synergistic viability effects in colorectal cancer models (A) A combination drug screen was performed with MEKi trametinib vs. 4 other top candidates interacting with radiation, derived from the radiosensitization screening experiments shown in [223]Figure 2 (PI3K inhibitor, PARP inhibitor, EGFR inhibitor, and CHK1 inhibitor) in matrices of 7 × 7 concentrations (8 × 8 including DMSO) using 7 organoid lines. (B) Synergy scores according to Bliss synergy, highest single agent (HSA), Loewe, and zero interaction potency (ZIP) models for the 4 drugs in combination with trametinib are shown. The overall scores represent the highest score of all dose combinations tested, 2 biological replicates were analyzed for D030T and D157T, 3 replicates were analyzed for D007T, D052T, D134T, and D160, and 4 replicates were analyzed for D080T. (C) Three-dimensional response (% inhibition) surface of the talazoparib and trametinib combination, exemplified by D134T organoids. The surface contains fitted values. (D) Bliss synergy surface of the talazoparib and trametinib combination in D134 organoids. The surface contains fitted values. (E) Heatmap of response (% inhibition) of trametinib-talazoparib combinations. The mean values of all seven tested organoid lines are shown; values for the individual lines were calculated as means of 2–4 biological replicates, as indicated in (B). Results of individual organoid lines are found in [224]Figure S12. (F) Heatmap Bliss synergy score of trametinib-talazoparib combinations. The average of bliss synergy scores for each dose combination of all seven tested organoid lines is shown. Results of individual organoid lines are found in [225]Figure S12. (G) Growth inhibition of 4 representative cancer organoid lines treated with increasing concentrations of trametinib in the presence of talazoparib at 0.039 and 0.16 μM. Expected response according to Bliss synergy model and observed response are shown. The mean values of 3 biological replicates are shown for D007T, D134T, and D160, and of 4 replicates for D080T. (H and I) Viability after combinatorial inhibition of MEK and PARP in short-term and long-term viability assay in CRC cell lines. SW480 (H) and DLD1 (I) were treated for 4 days with trametinib and talazoparib in a concentration matrix, followed by cell viability measurement. Results were normalized to the DMSO control. Means of 3 biological replicates are presented (left). Long-term colony formation assays showing the combinatorial inhibition of MEK and PARP on colony growth compared to any single reagent treatment in CRC cell lines (right). Representative scans of complete wells of standard 6-well plates are shown (9.6 cm^2 per well). See also [226]Figures S11 and [227]S12. Radiation synergizes with MEK-PARP combination therapy Having shown synergistic viability effects of MEKi with PARPi, we hypothesized that this drug combination would further synergize with radiation in CRC and allow low-dose application of both drugs in this setting, as both compounds target the DNA repair pathway. We therefore analyzed the effect of radiation on combinations of trametinib and talazoparib in cancer organoids using high-density drug concentration matrices, as described earlier. In seven tested organoid lines (including irradiation-resistant lines, lines with stronger irradiation response, and two previously untested lines added as unbiased set), we observed a strong increase in response to combinations of the two drugs when additional radiation was performed ([228]Figures 6A and [229]S13A). Applying a Bliss synergy model, we calculated the expected response to radiation added to combinations of trametinib and talazoparib and found that the observed responses exceeding the calculated Bliss response, particularly at lower doses ranging from 0.6 to 39 nM trametinib combined with 0.039–2.5 μM talazoparib ([230]Figure 6A). This proved the synergy of the two-drug combination with additional radiation. Of note, higher concentrations of the two-drug combination with radiation led to complete killing of almost all organoids, showing the high efficacy of this combination ([231]Figures 6A and [232]S13A). To also prove the synergy of added PARPi to the combination of MEKi with radiation, which we had shown to be synergistic above, we calculated a second Bliss model. This model considered trametinib-radiation and added talazoparib as an independent perturbation ([233]Figures 6B and 6C). The observed combination response showed higher potency than the predicted response according to the Bliss model, proving synergy between PARPi and MEKi-radiation ([234]Figure 6C). We also confirmed the markedly enhanced effect of the two-drug-radiation combination by long-term colony-forming assays and short-term viability assays in CRC cell lines ([235]Figures 6D, [236]S13B, and S13C). Figure 6. [237]Figure 6 [238]Open in a new tab Radiation synergizes with MEK-PARP combination therapy (A) Response/inhibition matrix derived from talazoparib-trametinib combinations, averaged over all seven tested organoid lines: non-irradiated, irradiated, and Bliss expected response, according to a model of added radiation to fixed combinations of trametinib and talazoparib, as well as Bliss excess (observed response − expected response). Data were normalized to non-irradiated DMSO controls. The lowest 5–6 concentrations tested are shown for each drug. (B) Dose-inhibition relationships of trametinib-radiation in combination with talazoparib treatments. Bliss expected response was calculated by using trametinib-radiation as one perturbation and adding talazoparib as second perturbation. D007T, D030T, D080T, D134T, and D160T are shown as representative examples of seven tested organoid lines. D007T, D030T, D080T, and D160T were irradiated with 4 Gy, D134T as a more radiation-sensitive line was irradiated with 2 Gy. Mean values of 2 biological replicates are shown for D030T, 3 replicates for D007T, D134T, and D160, and 4 replicates for D080T. (C) Bar plots of Bliss expected response vs. observed response in organoid lines treated with radiation, 0.625 μM talazoparib and 0.6–9.8 nM trametinib. Bliss expected response was calculated according to the same model as described in (B). Mean values of 2 biological replicates are shown for D030T and D157T, 3 replicates for D007T, D052T, D134T and D160, and 4 replicates for D080T. (D) Long-term colony formation assays of radiation in combination with MEK inhibition and PARP inhibition on colony growth compared to any single reagent treatment in CRC cell lines. Representative scans of complete wells of standard 6-well plates are shown (9.6 cm^2 per well). See also [239]Figures S11 and [240]S13. Radiation combined with MEK and PARP inhibition leads to significant tumor growth inhibition in vivo To validate these findings and to evaluate potential systemic adverse effects, we performed drug-drug-radiation combination treatments in two xenograft models of CRC. To this end, we performed subcutaneous engraftment of SW480 and DLD1 cell lines in immunocompromised, athymic BALB/c mice. Upon tumor engraftment and after a tumor volume of approximately 100 mm^3 was reached, mice were assigned to receive either a single dose of irradiation (4 Gy), irradiation plus MEKi or PARPi, or irradiation in combination with MEKi and PARPi. Both MEKi and PARPi were administered via oral gavage from day 1– to 4, including the day of irradiation for a total of 4 days (see scheme in [241]Figure 7A). Mice were sacrificed on day 27. Results of these mouse experiments showed that irradiation plus short-term MEKi could not significantly reduce tumor growth when compared to irradiation alone in DLD1 and SW480 xenografts, and irradiation plus PARPi could reduce tumor growth only in DLD1 xenografts ([242]Figures 7B–7G). When MEKi and PARPi were combined, the antiproliferative effect was stronger than with single combinations and significantly exceeded the efficacy of radiation or single drug-radiation combinations. Notably, this short-term drug-radiation combination treatment was well tolerated as it caused only small differences in weight compared to radiation combined with single drug treatment ([243]Figure 7H). Figure 7. [244]Figure 7 [245]Open in a new tab Radiation combined with MEK and PARP inhibition leads to significant tumor growth inhibition in vivo (A) Schematic overview of the in vivo experiments. (B–D) Tumor volume of DLD1 xenografts according to treatment condition over the course of the experiment (B) and at day 27 (C). (D) Images of the tumors (n = 5 per group) after sacrifice of mice, sorted by treatment condition. (E–G) Tumor volume of SW480 xenografts according to treatment condition over the course of the experiment (E) and at day 27 (F). (G) Images of the tumors (n = 5 per group) after sacrifice of mice, sorted by treatment condition. (H) Weight of tumor-bearing mice during the experiments, according to experimental conditions. Significant weight differences were observed for the MEKi-PARPi-radiation group versus the untreated and radiation only groups. (B–H) Two-way ANOVA with Tukey’s multiple comparisons test was used to test statistical significance. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (I) Putative mechanisms of interaction of MEK-PARP-radiation combination therapy. Radiation leads to DNA damage, which induces the cellular DNA damage repair machinery to enable cancer cell survival. Additionally, the RAS-MAPK pathway is upregulated. MEKi block radiation-induced RAS-MAPK signaling and downregulate RAD51, a core protein of the DNA DSB homologous recombination repair pathway. Addition of PARPi further enhances the effect by targeting DNA damage response via a different target. Together, these findings revealed a synergistic effect of radiation with MEKi and PARPi treatment. Mechanistically, this radiosensitizing effect was mediated by blocking radiation-induced RAS-MAPK signaling, as well as by inference with inhibition of DNA damage response through depletion and inhibition of RAD51 by MEKi and additional targeting of this pathway with PARP inhibition ([246]Figure 7I). Discussion Chemoradiation is the main therapy for locally advanced rectal cancers, but until now, strategies that specifically target altered signaling pathways in this tumor entity are not established. Our rectal cancer organoid assays can recapitulate clinical responses to chemoradiation similar to previous reports.[247]^11^,[248]^12^,[249]^13 Building on this, we exploited the predictive value of our cancer organoid translational platform to discover drug combinations to enhance radiation response. We performed large-scale radiation-drug screens using this platform and showed that targeting RAS-MAPK signaling by clinically approved MEKi resulted in enhanced response when combined with radiation therapy. Using different CRC models, we revealed that suppression of radiation-induced RAS-MAPK pathway activation and homology-directed DNA repair via RAD51 are major mechanisms by which MEK inhibition enhances radiotherapy. Finally, combinatorial drug-pair plus radiation experiments revealed that the effect of MEKi and radiation in rectal cancer models could be further increased by additional PARP inhibition, which was confirmed in vivo by murine xenograft models. Thus, our study provides strong experimental rationale to combine radiotherapy with two clinically approved targeted therapies as a treatment strategy for rectal cancers. A modulating effect of the RAS-MAPK pathway on radiosensitivity has been described in other cancer entities.[250]^27 MEKi were reported to boost the effect of radiation in pancreatic,[251]^28 lung,[252]^29 and mammary cancer cell lines.[253]^30 Several underlying mechanisms for the radiosensitizing effect of MEKi have been described, most involving DNA damage repair. These include suppression of homologous recombination genes such as DNA-PKcs in different tumor entities,[254]^28^,[255]^31 resulting, for instance, in a BRCA-like state in melanoma.[256]^32 According to our data, MEK inhibition does not affect the formation or resolution of DNA DSBs in CRC models, as observed in lung and pancreatic cancer cell lines.[257]^29 Instead, our findings suggest that RAD51, but not other important components of the DNA repair pathway, is downregulated in both CRC cell lines and organoids by MEK inhibition. RAD51 is a major component of the homologous recombination repair system and has been considered as a potential target to enhance radiosensitivity.[258]^33 RAD51 was also shown to be a marker of resistance to PARPi in BRCA-mutated breast cancer,[259]^34 and depletion of RAD51 via RNAi could re-sensitize cancer cells to PARP inhibition.[260]^35 A synergy between PARP and MEKi was observed in pancreatic and ovarian cancer, with mechanistic convergence on the homologous recombination repair pathway.[261]^36 Furthermore, a dual PARP-RAD51 inhibitor was developed[262]^37 and showed antineoplastic effects in the absence of radiation. These studies support our observation that downregulation of RAD51 is a potential mechanism for both the radiosensitizing effect of MEKi and its synergy with PARPi. We also observed irradiation-induced activation of RAS-MAPK signaling. RAS-MAPK signaling, as a proliferative signal in cancer cells, can play a role in radio-resistance by promoting tumor growth and overcoming cell-cycle arrest. Accordingly, MEKi abolished radiation-induced activation of RAS-MAPK signaling as an additional mechanism of radiosensitization and a putative way to overcome radio-resistance. Radiation-induced DNA damage can activate RAS-MAPK signaling in untransformed cells such as fibroblasts[263]^38 and keratinocytes,[264]^39 but also in pancreatic or breast cancer cell lines.[265]^40^,[266]^41 The underlying mechanisms described so far are manifold and include activation of ERK via GADD45β in breast cancer or stimulation of the pathway at the receptor levels via HER1/EGFR.[267]^42^,[268]^43^,[269]^44 ERK1/2 signaling is essential for activation of the G2/M cell cycle checkpoint in response to DNA damage by radiation[270]^45 and also associated with transcriptional upregulation of DNA repair genes, such as ERCC1 and XRCC1.[271]^46 Furthermore, radiation-induced ERK1/2 signaling can activate DNA-PKcs, which plays a critical role in non-homologous end joining-mediated DSB repair.[272]^31 These findings indicate that radiation-induced ERK signaling might represent a specific cellular adaptation to overcome DNA damage, which can be pharmacologically targeted. However, this effect of radiation was not observed in all of our investigated CRC models, indicating that this adaptive response may be subtype specific. Both MEKi and PARPi have been tested separately in early clinical studies as enhancers and sensitizers of radiation therapy in rectal cancers. A phase 1 trial has been conducted in patients with rectal cancer to determine the maximum tolerated dose of trametinib added to 5-FU-based chemoradiation.[273]^47 A pathological complete response rate of 25% was observed at the maximum tolerated dose, and the treatment was overall well tolerated. Due to the single-arm design, the radiosensitizing effect could only be estimated, but it was higher than the complete response rate of 15% of a matched historical cohort. PARPi can also increase radiosensitivity of CRC cells, particularly in the setting of XRCC deficiency.[274]^48 A phase 1 clinical trial assessed the maximum tolerated dose of the PARPi veliparib combined with neoadjuvant chemoradiation with capecitabine. The combination treatment was well tolerated with no dose-limiting grade III or IV adverse effects and achieved a pathological complete response rate of 28%.[275]^49 Future (clinical) studies may also reveal if a specific subgroup of rectal cancers can particularly benefit from adding MEK-PARP inhibition to radiation therapy. Specifically, whether MEKi-PARPi combinations will potentially increase the radiosensitizing effect of currently used chemotherapeutic agents such as 5-FU remains to be explored by clinical trials. Limitations of the study For screening experiments, we chose 2–4 Gy as the screening radiation doses. While varying radiation doses for all lines could potentially have yielded different results in drug-radiation combination screening experiments, preliminary experiments with 4 Gy revealed considerable radiation effects while not being lethal to most cells as a single fraction, thereby allowing a good dynamic range in our experiments leading to the identification of relevant candidate combinations. Additionally, 4 Gy represents a clinically relevant, single-fraction dose in the context of radiation research and is commonly used in preclinical studies.[276]^29^,[277]^31 Organoid lines with a strong intrinsic sensitivity to radiation were irradiated with a reduced dose of 2 Gy to allow a good dynamic range of drug-radiation combination tests. Our experimental results are based on different preclinical tumor models, including patient-derived organoids and murine xenograft models of common CRC cell lines. A major concern for clinical translation is the potential intestinal toxicity of the MEKi/PARPi-radiation combination. Although we observed only a minor weight loss in our murine xenograft models during combination treatment, the subcutaneous engraftment does not allow us to assess potential adverse effects on more radiation-sensitive tissues such as the intestinal mucosa adjacent to the tumor. Furthermore, resistance mechanisms to RAS-MAPK pathway inhibition could be mediated by the tumor microenvironment, including cancer-associated fibroblasts.[278]^50 These factors are not properly captured by our cancer organoid models, and therefore, the synergistic effect that we observed could be overestimated. Clinical trials will be needed to sufficiently address both questions of toxicity and efficacy of MEKi/PARPi-radiation combinations in rectal cancer. Lastly, the mechanism by which MEKi reduces RAD51 remains not exactly defined. Our experimental results suggest that mRNA levels of RAD51 are not reduced, and no active degradation of RAD51 was observed in the cycloheximide chase assays. Therefore, we suggest that future research should focus on translational regulation of RAD51 by MAPK-RAS signaling by using, for instance, polysome profiling. In conclusion, we used an organoid platform to discover a strong synergy effect of PARP-MEKi combination with radiotherapy in rectal cancer. We provide molecular explanations for the radiosensitizing effects of MEKi, indicating a convergence of the two inhibitors on the DNA repair pathway. Given that both PARPi and MEKi show promising results in phase 1 neoadjuvant radiation trials with low levels of toxicity, our study advocates combining both agents with radiation in future clinical trials for rectal cancer. Resource availability Lead contact Further information and requests for resources and reagents should be directed to the lead contact, Johannes Betge (j.betge@dkfz.de). Materials availability Requests for materials or reagents should be directed to the lead contact. Data and code availability * • Expression profiling data were deposited in Gene Expression Omnibus (GEO:[279]https://www.ncbi.nlm.nih.gov/geo/ with project number [280]GSE294953). Proteomics data were deposited in the PRIDE repository and are available in ProteomeXchange: PXD063024. Next-generation sequencing data of organoids can be made available through the European Genome Phenome Archive (EGA: [281]https://ega-archive.org under the accession number EGAD00001004313) and the German Human Genome Phenome Archive (GHGA: [282]https://data.ghga.de/ under the accession number GHGAS14639338878282). Data access requests for sequence data will be evaluated and transferred upon completion of a data transfer agreement and authorization by the data access committee at the University Medical Center Mannheim and DKFZ under the premise of adhering to EU General Data Protection Regulation. * • No nonstandard code was used to generate, analyze, or plot the data presented in this study. Codes to analyze the data and generate the plots are available from the corresponding authors upon reasonable request. * • Any additional information required to reanalyze the data reported in this work paper is available from the [283]lead contact upon reasonable request. Acknowledgments