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. 2015 Apr;5(4):358-67.
doi: 10.1158/2159-8290.CD-14-1518. Epub 2015 Feb 11.

Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations

Leanne G Ahronian  1 , Erin M Sennott  1 , Eliezer M Van Allen  2 , Nikhil Wagle  2 , Eunice L Kwak  1 , Jason E Faris  1 , Jason T Godfrey  3 , Koki Nishimura  3 , Kerry D Lynch  4 , Craig H Mermel  5 , Elizabeth L Lockerman  3 , Anuj Kalsy  3 , Joseph M Gurski Jr  1 , Samira Bahl  6 , Kristin Anderka  6 , Lisa M Green  6 , Niall J Lennon  6 , Tiffany G Huynh  4 , Mari Mino-Kenudson  4 , Gad Getz  5 , Dora Dias-Santagata  4 , A John Iafrate  4 , Jeffrey A Engelman  1 , Levi A Garraway  2 , Ryan B Corcoran  7
Affiliations

Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations

Leanne G Ahronian et al. Cancer Discov. 2015 Apr.

Abstract

BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance.

Significance: RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.

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Figures

Figure 1
Figure 1. KRAS mutation leads to resistance to combined RAF/EGFR and RAF/MEK inhibition
(A) Parental VACO432 cells (VACO) and derivatives made resistant to combined RAF/EGFR inhibition (VACO-RE) or RAF/MEK inhibition (VACO-RM) were treated for 3d with the indicated concentrations of vemurafenib (VEM) and cetuximab (CET) or vemurafenib and selumetinib (SEL). Relative cell titer was determined by Cell TiterGlo assay. (B) Sanger sequencing of exon 2 of KRAS from genomic DNA isolated from VACO, VACO-RE, and VACO-RM cells. (C) VACO432 cells expressing exogenous KRAS G12D, G13D, or empty vector control were treated as in (A) and relative cell titer was determined. (D) VACO432 cells expressing KRAS G12D, G13D, or empty vector control were treated for 24h with the indicated concentrations of drugs, and western blotting was performed with the indicated antibodies. (E) VACO432 cells expressing exogenous KRAS G12D, G13D, or empty vector control were treated with the ERK inhibitor VX-11e (VX) as in (A) and relative cell titer was determined. (F) Cells were treated as in (D) for 24h with the indicated concentrations of VX-11e and western blotting was performed with the indicated antibodies.
Figure 2
Figure 2. KRAS amplification can drive clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition
(A) Clinical time course of therapy for BRAF-mutant CRC patient #1 showing dates of therapy and timing of biopsies. Biopsies #1 (post-RAF/MEK) and #2 (post-RAF/EGFR) were taken from distinct metastatic lesions. Serum CEA tumor marker levels and cumulative tumor diameter as measured by RECIST are shown throughout the treatment course. (B) RECIST measurements of individual target lesions during therapy with encorafenib plus cetuximab from April 30 to September 23, 2013. Target lesion #3 was biopsied upon progression. (C) CT images of target lesion #3 (indicated by yellow arrow) throughout therapy with dabrafenib plus trametinib and encorafenib plus cetuximab. (D) DNA copy number traces for chromosome 12 showing focal amplification of KRAS in the post-RAF/EGFR biopsy. KRAS transcript abundance as determined by RNA-seq are also shown for each sample (RPKM = reads per kilobase of transcript per million mapped reads). (E) FISH was performed on biopsy specimens using probes for KRAS (red) and chromosome 12 (Chr12; green). (F) VACO432 cells overexpressing YFP control or wild-type KRAS (KRAS WT) were treated for 72h with the indicated concentrations of drug, and relative cell titer was determined. (G) VACO432 cells overexpressing YFP or KRAS WT were lysed, and western blotting was performed with the indicated antibodies. (H) Western blot of VACO432 cells overexpressing YFP or KRAS WT were treated with the indicated concentrations of drug for 24h.
Figure 3
Figure 3. BRAF amplification causes clinical acquired resistance to combined RAF/EGFR inhibition
(A) Clinical time course of therapy for BRAF-mutant CRC patient #2 showing dates of therapy and timing of post-progression biopsy. CEA tumor marker levels and cumulative tumor diameter as measured by RECIST are shown throughout the treatment course. (B) DNA copy number traces are shown for the post-progression biopsy compared to a pre-treatment biopsy taken from the same lesion immediately prior to the start of panitumumab + dabrafenib therapy. Focal amplification of BRAF on chromosome 7 is shown in the post-progression biopsy. BRAF V600E mutant allele frequencies are shown for each sample. (C) FISH was performed on the pre-treatment and post-progression biopsies using probes for BRAF (red) and chromosome 7 (Chr7; green). (D) VACO432 cells overexpressing BRAF V600E compared to empty vector were lysed, and western blotting was performed with the indicated antibodies. (E) Cells from (D) were treated for 72h with the indicated concentrations of dabrafenib (DAB), panitumumab (PAN), trametinib (TRA), or VX-11e (VX) for 72h and relative cell titer was determined. (F) Cells from (D) were treated for 24h with the indicated concentrations of drugs, and western blotting was performed with the indicated antibodies.
Figure 4
Figure 4. MEK1 F53L mutation drives clinical acquired resistance to combined RAF/MEK inhibition
(A) Clinical time course of therapy for BRAF-mutant CRC patient #3 showing dates of therapy and timing of post-progression biopsy. Cumulative tumor diameter as measured by RECIST is shown throughout the treatment course. (B) List of key mutations identified in the post-progression biopsy with associated allele frequencies. (C) Sanger sequencing of a patient-derived cell line generated from the post-progression biopsy for BRAF V600E, ARAF Q489L, and MEK1 F53L. All mutations were found to be present in 30 of 30 single cell clones. (D) Cells expressing the indicated constructs were treated with dabrafenib plus trametinib or VX-11e as shown for 72h, and relative cell titer was determined. (E) Cells expressing wild-type MEK1 (MEK1 WT), MEK1 F53L, or empty vector control were treated with the indicated concentrations of drugs for 24h, and western blotting was performed with the indicated antibodies.
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References

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