AACR 101st Annual Meeting 2010, Washington D.C. Experimental and Molecular Therapeutics Section 29; Abstract #3855

Transcription

1 Investigation of the Growth Inhibitory Activity of the MEK Inhibitor ARRY-162 in Combination with Everolimus in a Variety of KRas and PI3K Pathway Mutant Cancers Brian Tunquist, Tyler Risom, Debbie Anderson, Jennifer Garrus, Shannon Winski, Eli Wallace, Jim Winkler, Kevin Koch, Patrice Lee, Duncan Walker, and Stefan Gross AACR 101st Annual Meeting 2010, Washington D.C. Experimental and Molecular Therapeutics Section 29; Abstract #3855

2 Inhibition of the Raf/MEK/ERK Pathway in Oncology Oncogenic mutations of certain receptor tyrosine kinases (RTK), Ras, and Raf frequently manifest in constitutive activation of MEK1/2 and ERK1/2 kinases. This pathway has attracted much attention in the search for new chemotherapeutics, and small molecule inhibitors of MEK1/2 have been identified. ARRY-162 is a potent and selective inhibitor of MEK1/2: RTKs frequently mutated in cancers Ras Raf MEK1/2 ARRY-162 ERK1/2 Cell Proliferation

3 Inhibition of the RAF/MEK/ERK Pathway in Oncology Mutation of Raf, and to some extent, Ras, confers sensitivity to the MEK inhibitor AZD6244 (ARRY ). Mutational activation of the PI3K pathway correlates with resistance. Dry et al, Cancer Res, 2010

4 Ras Activation of PI3K and Raf Pathways in Cancer Ras activates the PI3K and Raf/MEK/Erk signaling pathway. The PI3K pathway may compensate for the loss of Raf/MEK/Erk signaling. RTKs Ras PTEN PI3K PDK1 AKT Crosstalk Raf MEK ERK ARRY-162 FOXO RSK Cell survival frequently mutated in cancers TORC1 S6K 4EBP1 Cell Growth, Metabolism Cell Proliferation, Angiogenesis

5 Inhibition of MEK and mtor Signaling Pathways Would inhibition of Raf/MEK/ERK and mtor signaling pathways yield greater activity in the clinic through enhanced disruption of survival and proliferation signals? RTKs Ras PTEN PI3K PDK1 AKT Crosstalk Raf MEK ERK ARRY-162 FOXO Everolimus RSK Cell survival frequently mutated in cancers TORC1 S6K 4EBP1 Cell Growth, Metabolism Cell Proliferation, Angiogenesis

6 Experimental Design Cell Lines: 47 in total encompassing a wide range of tumor types and mutational status Specifically, mutation of BRaf, Ras, PIK3CA, and PTEN genes were of interest. Assay: Proliferation (Alamar Blue, DNA), 3-5 day duration Compounds: Everolimus: 10 nm (minimum concentration yielding maximum growth inhibition across all lines) ARRY-162: 10, 30 & 100 nm (2x, 6x and 20x mechanistic cell IC 50 ) Combination Activity Determination: Bliss Method of Fractional Independence (Bliss, CI. Ann Appl Biol. 1939; 26: ) Combination Index Antagonism Independence Additivity Synergism

7 ARRY-162 and everolimus in vitro Combination Activity in Malme3M Cells 100 percent of control (POC) CI = 0.44 Combination Index Antagonism Independence/ Additivity Synergism 0 100nM ARRY nM everolimus combination percent of control (POC) [ARRY-162] (μm) [everolimus] (nm)

8 Tumor Cell Line Data Summary: 100 nm ARRY nm Everolimus Combination Tumor Cell Lines (grouped by genotype) 1.5 Antagonism Combination Index (CI) Independence/ Additivity Synergy WT Raf/Ras Mut Raf or Ras - Enhanced combinatorial activity in Ras or Raf mutant tumor cell lines: - majority of cell lines exhibit a CI value of less than 1 - many of the cell lines exhibit synergy (CI < 0.8)

9 Further Stratification Based Upon PI3K Pathway Mutation Tumor Cell Lines (grouped by genotype) 1.5 Antagonism Combination Index (CI) 1.0 Independence/ Additivity Synergy 0.5 WT Raf/Ras Mut Raf or Ras WT Raf/Ras Mut Raf or Ras WT Raf/Ras Mut Raf or Ras PIK3CA PTEN Mutation - Combination activity may not be enhanced with an additional mutation of PI3K, while tumors with a PTEN mutation may respond negatively. - Experiments analyzing the effect of PTEN knockdown in sensitive cell lines are underway.

10 Does in vitro combination activity translate in vivo? Tumor Cell Lines (grouped by genotype) 1.5 Antagonism Combination Index (CI) SW620 NCI-H460 Independence/ Additivity Synergy WT Raf/Ras Mut Raf or Ras - SW620 colorectal cancer tumor cell line: KRas (G12V), CI = NCI-H460 lung cancer tumor cell line: KRas (Q61H), PI3KCA (E545K), CI = 0.73

11 in vivo Comparison of SW620 and NCI-H460 Cell Lines SW620 NCI-H460 Tumor Volume (mm3) Day of Study Vehicle Everolimus, 2.5 mg/kg, QDx12 ARRY-162, 100 mg/kg, BIDx13 ARRY-162, 100 mg/kg, BIDx13 Everolimus, 2.5 mg/kg, QDx Day of Study Vehicle Everolimus, 2.5 mg/kg, QDx12 ARRY-162, 100 mg/kg, BIDx16 ARRY-162, 100 mg/kg, BIDx16 Everolimus, 1 mg/kg, QDx16 - in vitro combination activity is consistent with in vivo xenograft data. Deb Anderson/Shannon Winski

12 Pathway Inhibition Comparison SW620 NCI-H460 SW620 NCI-H460 ARRY-162 (100nM) Everolimus (10nM) ARRY-162 (100nM) Everolimus (10nM) Phospho-Erk1/2 (T202/Y204) Phospho-eIF 4EBP1 (Thr37/46) Erk1/2 Phospho-S6 (Ser235/236) Phospho-Akt (S473) PTEN LKB1/STK11 Phospho-Akt (T308) GAPDH Akt - Decreased perk levels by ARRY Unable to identify markers of sensitivity/resistance to the combination.

13 Profiling Apoptosis and Cell Cycle Regulators SW620 NCI-H460 SW620 NCI-H460 ARRY-162 (100nM) Everolimus (10nM) ARRY-162 (100nM) Everolimus (10nM) Bim p27 Kip1 Phospho-Rb (Ser608) Mcl-1 GAPDH Rb GAPDH - No change seen in levels of Bcl-2, Bcl-X L, Bid, NOXA, or cleaved PARP (not shown). - Regulation of Bim, p27 Kip1, and Rb proteins by MEK inhibitors has been reported. - Unable to identify markers of sensitivity/resistance to the combination.

14 Summary ARRY-162 is a potent and selective inhibitor of MEK1/2 Combination activity of ARRY-162 and everolimus may be enhanced by selection of tumors with specific genetic alterations: Mutation of Ras or Raf is a potential marker of additivity/synergy No clear impact from mutation of PI3K or PTEN Possible exception is PTEN in wildtype cell lines Investigations are underway to determine whether loss of PTEN is able to decrease combination activity in sensitive cells Combination activity in vitro correlates with activity in vivo Analysis of obvious pathway components has not revealed biomarkers of sensitivity/resistance to the combination thus far.

15 Preliminary Implications Patient Selection: Patients with mutations in Ras or Raf may benefit from ARRY-162/everolimus combination treatment Patients with mutation of PI3K may also benefit from this combination Patients with a loss of function mutation in PTEN may not respond to this combination Key indications with a high incidence of Ras or Raf mutations include: melanoma NSCLC colorectal cancer Cell Autonomous vs. Tumor Microenvironment Additional benefit may be gained from effects of this combination on tumor hypoxia and metabolism. ARRY-162 is currently in a Phase 1 study in patients with solid tumors.