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Purpose:Encorafenib + cetuximab (E+C) is an effective therapeutic option in chemorefractory BRAFV600E metastatic colorectal cancer (mCRC). However, there is a need to improve the efficacy of this molecular-targeted therapy and evaluate regimens suitable for untreated BRAFV600E in patients with mCRC.Experimental Design:We performed a series of in vivo studies using BRAFV600E mCRC tumor xenografts. Mice were randomized to receive 5-fluoruracil (5-FU), irinotecan, or oxaliplatin regimens (FOLFIRI or FOLFOX), (E+C) or the combination. Patients received long-term treatment until disease progression, with deescalation strategies used to mimic maintenance therapy. Transcriptomic changes after progression on cytotoxic chemotherapy or targeted therapy were assessed.Results:Antitumor activity of either FOLFIRI or E+C was better as first-line treatment as compared with second-line, with partial cross-resistance seen between a cytotoxic regimen and targeted therapy with an average 62% loss of efficacy for FOLFIRI after E+C and a 45% loss of efficacy of E+C after FOLFIRI (P < 0.001 for both). FOLFIRI-treated models had upregulation of epithelial–mesenchymal transition (EMT) and MAPK pathway activation, where E+C treated models had suppressed MAPK signaling. In contrast, with chemotherapy with E+C, EMT and MAPK signaling remained suppressed. FOLFOX or FOLFIRI, each in combination with E+C, were the most active first-line treatments as compared with E+C or to chemotherapy alone. Furthermore, FOLFOX in combination with E+C as first-line induction therapy, followed by E+C ± 5-FU as maintenance therapy, was the most effective strategy for long-term disease control.Conclusions:These results support the combination of cytotoxic chemotherapy and molecular-targeted therapy as a promising therapeutic approach in the first-line treatment of BRAFV600E mCRC.

Differentially enriched hallmark gene sets after cytotoxic chemotherapy and targeted therapy of B1003b PDX tumor samples.

Supplementary Table 3: EPHA2 expression in tumor samples with HSCORE

The EPHA2 tyrosine kinase receptor is implicated in tumor progression and targeted therapies resistance. We evaluated EPHA2 as a potential resistance marker to the antiepidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in colorectal cancer. We studied activation of EPHA2 in a panel of human colorectal cancer cell lines sensitive or resistant to anti-EGFR drugs. The in vitro and in vivo effects of ALW-II-41-27 (an EPHA2 inhibitor) and/or cetuximab treatment were tested. Formalin-fixed paraffin-embedded tumor specimens from 82 RAS wild-type (WT) metastatic colorectal cancer patients treated with FOLFIRI + cetuximab as first-line therapy in the CAPRI-GOIM trial were assessed for EPHA2 expression by immunohistochemistry and correlated with treatment efficacy. EPHA2 was differentially activated in colorectal cancer cell lines. Combined treatment with ALW-II-41-27 plus cetuximab reverted primary and acquired resistance to cetuximab, causing cell growth inhibition, inducing apoptosis and cell-cycle G1–G2 arrest. In tumor xenograft models, upon progression to cetuximab, ALW-II-41-27 addition significantly inhibited tumor growth. EPHA2 protein expression was detected in 55 of 82 tumor samples, frequently expressed in less-differentiated and left-sided tumors. High levels of EPHA2 significantly correlated with worse progression-free survival [8.6 months; confidence interval (CI) 95%, 6.4–10.8; vs. 12.3 months; CI 95%, 10.4–14.2; P = 0.03] and with increased progression rate (29% vs. 9%, P = 0.02). A specific EPHA2 inhibitor reverts in vitro and in vivo primary and acquired resistance to anti-EGFR therapy. EPHA2 levels are significantly associated with worse outcome in patients treated with FOLFIRI + cetuximab. These results highlight EPHA2 as a potential therapeutic target in metastatic colorectal cancer.

Supplementary Figure 1. A: Phosphoarray analysis of SW48 and SW48-CR cell lines. B: Silencing of EPHA2 decreases the sensitivity to ALW-II-41-27 in HCT15 and SW48-CR cell lines.

Supplementary Figure 2. A-B: Synergistic anti-proliferative effects of the combination of ALW-II-41-27 and cetuximab.C: Effect of Epha2 gene knockdown on cetuximab sensitivity in HCT116 and LOVO cell lines. D: Effects of combination treatment with ALW-II-41-27 and cetuximab on induction of apoptosis. E: Cell cycle distribution for HCT15 and SW48-CR following treatment with ALW-II-41-27 and/or cetuximab for 24, 48 and 72 hrs at indicated doses. F: Effects of EPHA2 blockade alone and in combination with cetuximab on intracellular signalling pathways of cell proliferation and survival.

Supplementary materials and methods

Supplementary Table 2: EPHA2 expression in tumor samples of 82 RAS WT colorectal cancer patients from the GOIM-CAPRI trial

Supplementary Table 1: Treatment with ALW-II-41-27 or cetuximab as single agents

supplementary figure 1. Effects of cetuximab in combination with regorafenib in a panel of CRC cell lines with primary and acquired resistance to anti-EGFR inhibitor in vitro.

supplementary table 1. mutational status

supplementary figure 3. Pro-apoptotic effects of cetuximab in combination with regorafenib in SW620 CRC cell line with primary resistance to anti-EGFR inhibitor.

supplementary figure legend

supplementary figure 4. Effects of cetuximab in combination with regorafenib on mice body weight.

Purpose: The use of cetuximab in the treatment of metastatic colorectal cancer is limited by development of resistance. Experimental Design: We have investigated in three models of highly epidermal growth factor receptor (EGFR)–dependent colorectal cancer xenografts, the effect of maintenance therapy with different kinase inhibitors alone or in combination with cetuximab, after cytotoxic treatment induction with irinotecan plus cetuximab. Results: SW48, LIM 1215, and GEO colorectal cancer cell lines were engrafted into nude mice and treated for 3 weeks with irinotecan and/or cetuximab. The combined treatment induced a significant reduction of tumor size. A subsequent experiment was performed in all three xenograft models in which after an induction treatment with irinotecan plus cetuximab, mice were randomly assigned to one of the following treatments: control, cetuximab, regorafenib, a selective PIK3CA inhibitor (PIK3CAi), a selective MEK inhibitor (MEKi), and/or the combination of each inhibitor with cetuximab. The cetuximab plus MEKi treatment determined the best antitumor activity with suppression of tumor growth. This effect was prolonged for 13 to 15 weeks after cessation of therapy and was accompanied by prolonged survival. Antitumor activity was accompanied by inhibition of the MAPK and MEK pathways. Moreover, in the cetuximab plus MEKi-treated SW48 xenograft group, KRAS mutations as a mechanism of acquired resistance were detected in 25% of cases compared with 75% KRAS mutations in the MEKi-treated group. Conclusions: A possible strategy to prevent and/or overcome resistance to anti-EGFR inhibitors in metastatic colorectal cancer is a maintenance therapy with cetuximab plus MEKi after an initial treatment with irinotecan plus cetuximab. Clin Cancer Res; 21(18); 4153–64. ©2015 AACR.

DNA damage response (DDR) is crucial in a variety of tumors and several new drugs that interfere with this mechanism are already available or in advanced clinical testing. RAS-MAPK pathway activation, induced by either RAS or BRAF mutation, is a frequent feature of colorectal cancers (CRCs) and is strongly associated to mitotic stress and dependency upon DDR. Among the drugs used for CRC, oxaliplatin and irinotecan are known to induce DNA damage in form of single or double strand breaks (SSB/DSBs) and thus require active DDR systems in cancer cells to be tolerated. The poly-ADP-Ribose Polymerase (PARP) inhibitor Niraparib (MK-4827) is an FDA-approved orally bioavailable drug with strong PARP1-trapping activity, a feature ofted associated with synergism in combination with platinum salts, while synergism with other DNA damaging agents is independent of the PARP1-trapping activity. We tested the activity of niraparib used in combination with oxaliplatin or irinotecan (in the form of its active metabolite SN38) in a panel of 8 KRAS or BRAF mutated CRC cell lines (LOVO, HCT15, SW1116, LS1034, SW948, WiDr, SW480, HCT116) with different microsatellite and CIMP status. Combination index (CI) analysis was performed in order to evaluate the synergism between niraparib and the chemotherapeutics. Cell cycle distribution and apoptosis assays were performed in order to further characterize the effects of the combinations. The combination between niraparib and oxaliplatin resulted synergistic in 6 out of 8 cell lines, with strong synergism (CI < 0,5) in 2 cell lines, while the combination between niraparib and irinotecan (SN38) resulted synergistic in 7 out of 8 cell lines, with strong synergism in 5 cell lines and very strong synergism (CI < 0,1) in 3 cell lines. Interestingly, the only non-synergistic cell line to the combination with irinotecan is also non-synergistic to the combination with oxaliplatin; on the other hand, the cell lines that exhibit strong synergism to the combination with irinotecan do not present strong synergism to the combination with oxaliplatin and vice versa. Moreover, all the microsatellite instable (MSI) cell lines tested resulted synergistic for the two combinations. Finally, the synergistic combinations showed an increased induction of apoptosis and cell cycle arrest compared to non-synergistic combinations. Taken together, these data investigate the combination between the PARP inhibitor niraparib and the antiproliferative agents oxaliplatin and irinotecan in KRAS/BRAF mutated CRC cell lines, showing how MSI status predicts good synergism with the drug combinations, while MSS status is not a good predictor of synergism. Further studies are needed to identify better predictive biomarker beyond MSI status, in order to optimize the use of these combinations for future development. Citation Format: Pietro Paolo Vitiello, Davide Ciardiello, Claudia Cardone, Giulia Martini, Valentina Belli, Nunzia Matrone, Luca Poliero, Carola Borrelli, Pasquale Vitale, Nicoletta Zanaletti, Teresa Troiani, Davide Melisi, Fortunato Ciardiello, Erika Martinelli. Synergism between oxaliplatin or irinotecan with the PARP inhibitor niraparib in a preclinical model of KRAS/BRAF mutated colorectal cancer is associated with MSI status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 295.

According to the consensus molecular subtypes (CMS), almost 23% of human colorectal cancer (CRC) are classified in the CMS4 group, characterized by a mesenchymal signature and the activation of transforming growth factor β (TGF-β) signaling. AXL is a tyrosine kinase receptor involved in epithelial to mesenchymal transition, angiogenesis and immune modulation in CRC. We have previously demonstrated that AXL gene is amplified in approximately 5% of human CRC, and that AXL inhibition causes a significant blockade of CRC cell proliferation and migration. Here we have evaluated the role of TGF-β signaling and the potential interaction between TGF-β and AXL in human CRC cell lines. We assessed the expression and activation of TGF-β and AXL in a panel of six human CRC cell lines by western blot (WB) and real time PCR. We tested the sensitivity of HCT116 and LOVO cells to treatment with galunisertib ( LY21209761), a selective TGF-β R1 inhibitor, by MTT, cell invasion, wound healing and soft-agar colony forming assays. Further, intracellular CRC cell signaling was evaluated following galunisertib treatment by WB. TGF-β receptors 1 and 2 were expressed in all human CRC cell lines (HCT116, SW480, LOVO, LIM 1215, SW48 and CaCo2), whereas AXL expression was restricted to HCT116, SW480, LOVO cells. Treatment with galunisertib had little or no effect on cancer cell growth, whereas it partially reduced TGF-β induced cell migration, invasion and colony formation in HCT116 and LOVO cells (that co-expressed both TGF-β receptors and AXL). Interestingly, TGF-β inhibition resulted in a concomitant significant activation of AXL and p38 MAPK in both cell lines, that could represent a cancer cell adaptive mechanism of resistance to galunisertib treatment. These results suggest a potential functional cross talk between TGF-β, AXL and p38 MAPK signals in human CRC cells. In this respect, further experiments are ongoing to better understand this interaction with the aim of identifying potential novel anti-AXL and anti-TGF-β therapeutic strategies. Citation Format: Davide Ciardiello, Pietro Paolo Vitiello, Nunzia Matrone, Valentina Belli, Claudia Cardone, Luca Poliero, Carola Borrelli, Gianluca Arrichiello, Giulia Martini, Vincenza Ciaramella, Giusi Barra, Floriana Morgillo, Teresa Troiani, Davide Melisi, Fortunato Ciardiello, Erika Martinelli. Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2627.