Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms

Abstract

Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAF(V600E) models showed acquired drug resistance, one BRAF(V600E) model had a complete and durable response, and a BRAF(V600V) model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.

Keywords: BRAF; MEK; Melanoma; Vemurafenib; drug resistance; patient derived xenografts (PDX).

Figure 1

BRAF^V600E PDX tumor responses to Vemurafenib treatment reveal multiple resistance mechanisms. Mice were treated with either vehicle control or Vemurafenib (50 mg/kg orally, twice daily, 5 d on, 2 d off) for up to 150 days. The structure of Vemurafenib is displayed inside panel A. All vehicle treated tumors from patients 1-3 (green lines - ≥ n=4) rapidly increased in size. Chronic exposure to Vemurafenib (red line, except in panel B) produced differing responses among these PDX models. Y-axis values represent the mean tumor volume ± SEM (except for panel B) for each treatment group. A. Patient 1 tumors showed a significant, uniform response to Vemurafenib (n=11 started on treatment) with tumor growth inhibited for 50 d, after which acquired resistance emerged in all mice. Sampling of whole-tumor sections revealed progressively increased pERK expression by IHC staining (lower left panel) during treatment (day 50) and after resistance (day 70), with inter- and intra-tumor heterogeneity. Alternative splicing of the BRAF ^V600E gene was found in all four resistant tumors, but not in control vehicle-treated tumors or in A375 cells. Note that the dominant isoform is the 1.1 kb isoform (upper right, single asterisk), not the the 1.7 kb isoform (two asterisks). B. Patient 2 tumor growth is plotted for each individual mouse due to at the heterogeneous response to treatment with Vemurafenib (n=12 started on treatment). Resistant tumors with NRAS^Q61K mutations (frequency 33-50%) were present in three of four tumors examined; one resistant tumor lacked this point mutation, but had an alternatively spliced BRAF^V600E isoform of 1.1 kb. C. Patient 3 tumors showed a significant, uniform response to Vemurafenib (n=13 started on treatment) with tumor growth inhibited for 20 d, after which resistance emerged in all mice. No alternative BRAF splicing or NRAS mutations were observed. Three of the resistant tumors had elevated COT expression (lower right panel) relative to vehicle-treated tumors and to tumors isolated from patient 1. D. Patient 4 tumors displaying a reduction in tumor volume in response to chronic Vemurafenib treatment (n=12 started on treatment) did not develop resistance over the course of the study (80 days). Y-axis values represent the mean tumor volume ± SEM for each treatment group. The fidelity of the PDX model system is confirmed by the patient 5 BRAF^V600V tumors which did not respond by growth inhibition to either vehicle (n=5) or Vemurafenib (n=8). Y-axis values represent the mean tumor volume ± SEM for each treatment group.

Figure 2

BRAF gene expression and copy number in BRAF^V600E PDX models. BRAF gene expression (left axis black columns) and BRAF copy number (right axis, open columns) for PDX tumors versus parental tumors and normal melanocytes as controls. No consistent trends were identified.

Figure 3

MEK inhibition using PD0325901 inhibits Vemurafenib-resistant tumor growth in patient 1 but not patient 3 tumors. A. Vemurafenib-resistant grew to about 1000 mm³ during Vemurafenib treatment (red line) at which point PD0325901 (25 mg/kg orally, twice daily, 5 days on, 2 days off) triggersed a rapid reduction in tumor size over 14 days of co-treatment (purple line, n=3). B. High power magnification of Vemurafenib-resistant tumors exposed to PD0325901 plus Vemurafenib for 14 d (top image) reveals paucicellular fibrovascular tracks surrounding islands of viable melanoma cells with condensed nuclear chromatin and no nucleoli (H&E stain). Occasional nuclei are IHC-positive for the proliferation-associated marker Ki-67 (bottom left image); scattered apoptotic cells expressing activated caspase 3 were IHC positive (bottom right image). C. Stained whole mounts using (IHC followed by hematoxylin staining) show that both pERK and Ki-67 levels are reduced after Vemurafenib plus PD0325901 treatment. Scale bars indicate 1 mm. D. Vemurafenib-resistant tumors from patient 3 having elevated COT levels were transferred into new recipient mice and were exposed either to Vemurafenib alone (green line, n=5), or Vemurafenib plus PD0325901 (red line, n=6). The asterisk indicates the removal of mice from the study due to large tumor volumes.

Figure 4

Prolonged tumor size reduction of Vemurafenib-resistant tumors by once-daily dosing of PD0325901. A. Lowering the twice-daily dose of PD0325901 to once per day (25 mg/kg orally, Monday -Friday) sufficesd to maintain tumor suppression in Vemurafenib-resistant tumors; all tumor cells were eradicated in 3 of 4 tumors which had PD0325901 removed on day 70 and which were examined on day 100. B. When Vemurafenib-resistant tumors (500 mm³) were treated with PD0325901 (25 mg/kg orally, twice a day) their size decreased, and remained so until PD0325901 was removed, at which time there was rapid re-growth of the tumors.