PURPOSE Acquired resistance to anti-EGFR therapy (EGFRi) in CRC has previously been explained by the model of acquiring new mutations in KRAS/NRAS/EGFR, among other MAPK-pathway members. However, this was primarily based on single-agent EGFRi trials and little is known about the resistance mechanisms of EGFRi combined with effective cytotoxic chemotherapy in previously untreated patients. METHODS We analyzed paired plasma samples from RAS/BRAF/EGFR wild-type mCRC patients enrolled in three large randomized trials evaluating EGFRi in the first-line in combination with chemotherapy and as a single-agent in third-line. The mutational signature of the alterations acquired with therapy was evaluated. CRC cell lines with resistance to cetuximab, FOLFOX, and SN38 were developed, and transcriptional changes profiled. RESULTS Patients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared to those treated in combination with cytotoxic chemotherapy (9%). Further, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition (EMT). In contrast, commonly acquired resistance alterations in the MAPK pathway do not impact sensitivity to cytotoxic chemotherapy. CONCLUSION These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

KRAS and NRAS mutations occur in 45% of colorectal cancers (CRC), with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in metastatic CRC patients with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in CRC, along with the utility of PDX-based co-clinical trial platforms for drug development.

Designing studies of immunotherapy is limited due to a lack of pre-clinical models that reliably predict effective immunotherapy responses. To address this gap, we developed humanized mouse models of colorectal cancer (CRC) incorporating patient-derived xenografts (PDX) with human peripheral blood mononuclear cells (PBMC). Humanized mice with CRC PDXs were generated via engraftment of autologous (isolated from the same patients as the PDXs) or allogeneic (isolated from healthy donors) PBMCs. Human T cells were detected in mouse blood, tissues, and infiltrated the implanted PDXs. The inclusion of anti-PD-1 therapy revealed that tumor responses in autologous but not allogeneic models were more comparable to that of patients. An overall non-specific graft-vs-tumor effect occurred in allogeneic models and negatively correlated with that seen in patients. In contrast, autologous humanized mice more accurately correlated with treatment outcomes by engaging pre-existing tumor specific T-cell populations. As autologous T cells appear to be the major drivers of tumor response thus, autologous humanized mice may serve as models at predicting treatment outcomes in pre-clinical settings for therapies reliant on pre-existing tumor specific T-cell populations.

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

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

Purpose: Encorafenib plus cetuximab is an effective therapeutic option in chemorefractory BRAFV600E mCRC. However, there is a need to improve the efficacy of this molecular targeted therapy and evaluate regimens suitable for untreated BRAFV600E mCRC patients. Experimental Design: We performed a series of in vivo studies using BRAFV600EmCRC tumor xenografts. Mice were randomized to receive: 5-fluoruracil, irinotecan or oxaliplatin regimens (FOLFIRI or FOLFOX), encorafenib plus cetuximab (E+C) or the combination. Treatments included long term treatment until progression, with de-escalation strategies replicating maintenance treatments. Transcriptomic changes after progression on cytotoxic chemotherapy or targeted therapy were assessed. Results: Antitumor activity of either FOLFIRI or E+C was better in first-line as compared to second-line, with partial cross-resistance seen between cytotoxic regimen and targeted therapy with average 62% loss of efficacy for FOLFIRI after E+C and 45% loss of efficacy of E+C after FOLFIRI (p&lt;0.001 for both). FOLFIRI treated models had upregulation of 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 to E+C or to chemotherapy alone. Further, 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.

145 Background: Based on promising results from latest trials, a crucial point is to evaluate whether encorafenib (E) plus cetuximab (C), alone or in combination with chemotherapy, can improve clinical outcomes relative to current standard of care in previously untreated BRAFV600E mutant mCRC. Considering the high number of BRAFV600E mutant mCRC patients who will never receive a second-line treatment, the rationale of this strategy is to maximize treatment outcome within the first-line setting. Methods: We performed an in vivo study using human BRAFV600E CRC cell line-derived xenografts in nude mice. We evaluated the efficacy of encorafenib (E) + cetuximab (C), FOLFOX, and FOLFIRI, both as individual regimens and in combinations. Mice were treated for 3 weeks and followed for an additional 8 weeks to evaluate durability of tumor control. Additionally, we validated our findings using 3 BRAFV600E mutated patient derived xenografts. Tumors progressing on single agent and combined treatment were profiled by RNA sequencing, protein extraction for RPPA/Western blot, and establishment of in vitro primary cell cultures for further analyses. Results: Our study showed across all 4 models both FOLFOX and FOLFIRI, each in combination with encorafenib plus cetuximab, having greater efficacy than encorafenib plus cetuximab or either chemotherapy alone. No significant change in toxicity was seen with the addition of chemotherapy. Interestingly, in the one model with long term treatment, FOLFOX + E +/- C performed greatest over the long-term, with significant endpoint separation against all other treatment arms (P < 0.05). Conclusions: Taken together, results from our study suggest that the addition of chemotherapy to BRAF+EGFR targeted therapy can further increase the magnitude of response in BRAFV600E mCRC and is a promising combination now being explored clinically. Additionally, this research will substantially contribute to our understanding of the genetic and molecular bases of resistance to target therapies and chemo-based approach in BRAFV600Econtext.

<div>AbstractPurpose:<p>Encorafenib + cetuximab (E+C) is an effective therapeutic option in chemorefractory <i>BRAF</i>^V600E metastatic colorectal cancer (mCRC). However, there is a need to improve the efficacy of this molecular-targeted therapy and evaluate regimens suitable for untreated <i>BRAF</i>^V600E in patients with mCRC.</p>Experimental Design:<p>We performed a series of <i>in vivo</i> studies using <i>BRAF</i>^V600E 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.</p>Results:<p>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 (<i>P</i> < 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.</p>Conclusions:<p>These results support the combination of cytotoxic chemotherapy and molecular-targeted therapy as a promising therapeutic approach in the first-line treatment of <i>BRAF</i>^V600E mCRC.</p></div>