Conclusion ● The combination effect of BGB-283 and MEKi was positively

correlated to pMEK activation levels induced by MEKi as single agent (Figure 1,2). This data was consistent with previous findings that disruption of RAF-mediated MEK reactivation is required for effective inhibition of MAPK pathway in KRAS mutant tumors (Piro Lito, et al., Cancer Cell. 2014).

● Addition of BGB-283 in the presence of selumetinib (AZD6244) potently inhibited RAF-dependent MEK phosphorylation and led to sustained inhibition of ERK activity.

● BGB-283 and selumetinib showed synergistic effect in inhibiting proliferation of several RAS mutant NSCLC cell lines and exhibited enhanced antitumor activity in K-RAS mutant NSCLC and CRC xenograft models compared to monotherapies.

● Combining RAF inhibitor BGB-283 and MEKi could be a promising strategy in treating RAS mutated cancers.

Figure 5. (A) Combination of AZD6244 and BGB-283 demonstrated enhanced antitumor efficacy than each single agent alone and achieved 88% to 100% overall response rate (PR+CR) in Calu-6 xenograft model. (B) Combining BGB-283 and AZD6244 enhanced the antitumor activities of both compounds and achieved >100% TGI and 88% PR in HCT116 (KRASG13D) CRC xenograft model. (C) Pharmacodynamics (PD) studies conducted in Calu-6 xenograft model showed that combination of AZD-6244 with increased doses of BGB-283 significantly inhibited ERK phosphorylation. (*, p<0.05 vs. vehicle control; ^, p<0.05 vs. AZD6244 single treatment).

BGB-283 Effectively Enhances MEK Inhibitor Induced Tumor Suppression in RAS Mutant Cancers

Xi Yuan1, Zhiyu Tang1, Rong Du1, Shing-Hu Cheung1, Jing Wei1, Yuan Zhao1, Yunguang Du1, Rui Hao1, Xiaoxia Hu1, Wenfeng Gong1, Yong Liu1, Yajuan Gao1, Min Wei1,*, Changyou Zhou1, Lai Wang1, Lusong Luo1

1BeiGene (Beijing) Co., Ltd., Beijing 102206, China; *Correspondence: Min.Wei@beigene.com

Abstract #669

Introduction Mutations in K-RAS, which drives constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway, is one of the most frequent genetic alterations in human cancers. Pharmacological targeting RAS mutants directly is extremely challenging and targeted inhibition of RAS downstream effector MEK has shown limited clinical activity in suppressing the progression of RAS mutant tumors. The reduced sensitivity of RAS-mutated cancer cells to MEK inhibitors (MEKi) is associated with feedback phosphorylation of MEK and rebound of phosphorylated ERK levels after prolonged treatment. Three-tiered kinase module of RAF/MEK/ERK is integrated with negative feedback loops and generates a biological circuit serving as a negative feedback amplifier (NFA). Such NFA confers the robustness of the signalling pathway and makes it difficult to effectively inhibit the mediators within the circuit (Sturm OE, et al., Sci Signal. 2010). It is suggested that combining therapeutics that block multiple nodes within the feedback loops could disable the NFA function. In this study, we showed that combining BGB-283, a second generation BRAF inhibitor (Abstract #4692), and a series of MEKi could achieve better therapeutic effect of the latter.

Figure 2. The effect of different MEKis on the MEK/ERK pathway was investigated in Calu-6 cells using (A) western blot and (B) HTRF assay. AZD6244, PD0325901, and AS703026 treatment effectively reduced pERK levels but stimulated a robust MEK phosphorylation through feedback mediated by RAFs activation. In contrast, GSK1120212 and RO5126766 had minimal effect on MEK phosphorylation.

BGB-283 but not PLX4032 potently inhibits AZD6244-induced pMEK upregulation

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Figure 3. (A) BGB-283 inhibited AZD6244-induced MEK phosphorylation in Calu-6 cells with IC50 of 2.9 μM, 0.9 μM, and 0.6 μM for 1, 6, and 24 h treatment, respectively. In contrast, PLX4032 (vemurafenib) not only failed to inhibit but further induced MEK phosphorylation under the same conditions. (1 μM AZD6244 induced pMEK level was set as 100%.) (B) Combination of BGB-283 and AZD-6244 treatment led to sustained inhibition of both MEK and ERK phosphorylation for up to 48 h.

BGB-283 but not PLX4032 potentiates the inhibitory effect of AZD6244 on proliferation of Cau-6 cells

Figure 4. 1 μM BGB-283 and 1 μM AZD6244 as single agent showed partial inhibition on proliferation of Calu-6 cells. Combined treatment with BGB-283 and AZD6244 effectively blocked cell proliferation, which was concomitant with sustained inhibition of pMEK/pERK (Figure 2B). No such effect was observed when cells were treated with AZD6244 in combination of PLX4032.

Table 1. Synergistic effect was observed in combination of BGB-283 and AZD6244 (Selumetinib) in RAS mutant NSCLC cell lines.

Cell Line Mutations Number of Cells with

EOHSA

Average EOHSA per

Cell

Max EC50 Shift for

Selumetinib * Calu-6 KRAS, p53 43 (77%) 22 28  fold  ↓ A549 KRAS, CDKN2A 23 (85%) 11 5  fold  ↓

NCI-H2122 KRAS, p53, CDKN2A 28 (50%) 13 16  fold  ↓ NCI-H23 KRAS, p53, STK11 45 (80%) 18 10  fold  ↓

SW1573 KRAS, PIK3CA, CDKN2A, NF2 47 (83%) 18 64  fold  ↓

Sk-Lu-1 KRAS, p53, CDKN2A 43 (77%) 10 51  fold  ↓ Calu-1 KRAS 18 (67%) 4 N/A

NCI-H1299 NRAS 12 (44%) 6 N/A NCI-H358 KRAS 14 (52%) 6 N/A

The synergistic effect of BGB-283 and AZD6244 on cell proliferation was assessed in RAS mutant cells using EOHSA analysis. Various doses of BGB-283 were combined across a range of concentrations of AZD6244 to generate 9 × 9 matrix (Figure 1). Each combination was then scored to identify anti-proliferative effects that were greater than the effect of each individual component.

Table 2. No significant synergy was detected in combination of PLX4032 and AZD6244 in K/N-RAS mutant cells.

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Selumetinib* Calu-6 KRAS, p53 33 (57%) 9 1  fold    ↓ A549 KRAS, CDKN2A 8 (30%) 3 2  fold  ↑

NCI-H2122 KRAS, p53, CDKN2A 8 (14%) 7 25  fold  ↑ NCI-H23 KRAS, p53, STK11 45 (80%) 7 1  fold  ↓

SW1573 KRAS, PIK3CA, CDKN2A, NF2 11% 3 N/A

Sk-Lu-1 KRAS, p53, CDKN2A 51 (91%) 7 43  fold  ↓ Calu-1 KRAS 7 (26%) 3 N/A

NCI-H1299 NRAS 6 (22%) 3 N/A NCI-H358 KRAS 5 (19%) 1 N/A

*  Combinations  with  EOHSA  ≥  10  &  Max  EC50  shift  ≥  5  are  considered  to  have  synergy, highlighted in green

Combination of BGB-283 and AZD6244 shows improved efficacy in KRAS mutant xenograft models

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Figure 1. Heat maps showed that effect of combining BGB-283 and (A) AZD6244, (B) PD0325901, (C) AS703026, (D) GSK1120212, and (E) RO5126766 was evaluated by EHOSA analysis for percent inhibition of cell proliferation. BGB-283 effectively increased AZD6244, PD0325901 and AS703026 activity in Calu-6 cells (KRASQ61K). However, no obvious synergy was detected in combing BGB-283 with GSK1120212 or RO5126766.

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Differential combination effect of BGB-283 with MEKis is correlated with pMEK feedback levels

BGB-283 but not PLX4032 synergizes with AZD6244 in inhibiting RAS mutant cell proliferation

1 µM AZD6244+PLX4032 1 µM AZD6244+BGB-283