A personalized platform identifies trametinib plus zoledronate for a patient with KRAS-mutant metastatic colorectal cancer

Abstract

Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor's genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease's genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer.

Fig. 1. Overview and construction of Drosophila patient model.

(A) An outline of our approach. First, a comprehensive genomic analysis of the patient’s tumor and normal DNA [copy number, whole-exome sequencing (WES), and targeted HotSpot panel] was performed. Then, a personalized Drosophila model that captures a portion of the patient’s tumor’s genomic complexity was generated by targeting each Drosophila ortholog specifically in the Drosophila hindgut. After the model was validated, a high-throughput “rescue from lethality” drug screen was performed on FDA-approved drugs as single agents and in combination. Findings were then presented to a multidisciplinary tumor board. A personalized treatment plan based on the multidisciplinary tumor board’s recommendation was prepared and institutional review board–approved, followed by patient treatment. (B) Patient’s genomic landscape: Genes altered in the patient’s tumor, their functions, and Drosophila orthologs are indicated. LOH, copy number neutral loss of heterozygosity. MAPK, mitogen-activated protein kinase. (C) GAL4/UAS system used for targeted genetic manipulations in Drosophila. Transgenes targeting nine genes (ras85D^G12V, etc.) were cloned downstream of a GAL4-responsive UAS promoter and transgenic flies generated. Transgene expression was then induced in a tissue-specific manner by crossing transgenic flies to byn-gal4 for hindgut epithelium and to tubulin-gal4 for ubiquitous expression. GFP, green fluorescent protein. (D) Personalized construct generated for the patient, targeting nine genes. This construct expressed a GAL4-inducible (i) UAS-ras85D^G12V transgene and (ii) synthetic eight-hairpin cluster targeting the Drosophila orthologs of the eight tumor suppressor genes. After transgenic flies were generated, transgenic constructs UAS-ago^RNAi and UAS-apc^RNAi were genetically introduced by standard genetic crosses to increase overall ago and apc knockdown.

Fig. 2. Validating and screening Drosophila patient model.

(A) Expressing byn > GFP in control animals highlighted the hindgut in bright-field (top panels) and expression of the byn-GAL4 driver specific to the hindgut (bottom panels). Microscope magnifications (5× and 10×) are shown. (B) Expressing the 006.1 transgene set in the hindgut led to strong expansion of the anterior hindgut. The midgut/hindgut (M/H) boundaries are indicated; the dark regions in the 006 bright-field images likely reflect cell death. Image contrast was enhanced equally using Preview software for clarity. Scale bars, 100 μm. (C and D) Trametinib in combination with ibandronate or zoledronate rescued the lethality observed by the patient’s personalized Drosophila model. Concentrations indicate final food concentrations. Each data point represents a replicate with 10 to 15 experimental and 20 to 30 control animals. Raw numbers are provided in table S2C. Error bars indicate SEM. DMSO, dimethyl sulfoxide.

Fig. 3. Secondary assays of drug response.

(A) Western blot analysis of MAPK signaling pathway output from control and drug-treated hindgut lysates using dpERK as a readout. Quantification represents two independent experiments with different sets of biological replicates. Each experiment was performed in triplicate with 10 hindguts per biological replicate (gel images are shown in fig. S2C). (B and C) Analysis of the expansion of the anterior hindgut in control and drug-treated animals. (B) Quantification of the anterior region of the hindgut. Data points indicate individual hindguts. (C) Two images representing the high and low ends of the size distribution observed in the assay. Quantified region of the hindgut is outlined by white dashed lines. T, 1 μM trametinib; Z, 0.7 μM zoledronate in the food. Statistical significance in (A) and (B) was determined using multiple t tests with Holm-Sidak correction for multiple hypotheses.

Fig. 4. Patient response.

(A) Patient scans before treatment and 27 weeks after treatment. Arrow indicates an example of lesion in left supraclavicular node. (B) Two examples of target lesion shrinkage at indicated time points are highlighted by pink shading plus red dashed outline; the top panels provide detail to (A).