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The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader

CCR Translation for This Article from Dual Blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) Pathways Synergistically Inhibits Rhabdomyosarcoma Cell Growth In Vitro and In Vivo

Supplementary Table & Figure Legends - PDF file 41K, Detailed captions for: Suppl Table S1. Immunohistochemical analysis indicates correlation between pAKT(Ser473),pERK1/2(Thr202/Tyr204) and pS6(Ser235/236) in primary tumours Fig S1. High level expression of p110alpha in two of four ARMS cell lines. Fig S2. PIK3CA KD does not induce cell cycle arrest or apoptosis in the growth inhibited RMS cells. Fig S3. Downregulation of PTEN expression appears to be associated with delayed inhibition of AKT phosphorylation in RH30 cells following p110alpha KD. Fig S4 Increased sensitivity of the RMS-1 PIK3CA KD line to TGX221, and of the RMS-1, RD and RMS-YM KD lines to ZSTK474. Fig S5 Equivalent changes in the biomarkers of MAPK and PI3K activity at 6h in vivo, to those seen in vitro following acute dosing with AZD6244 and AZD8055 alone, and in combination, with recovery to control levels correlating with plasma and tumor clearance of both drugs. Fig S6 Tolerability study of twice daily dosing of 10mk/kg po AZD6244 and AZD8055 indicates reversible weight loss with combination treatment, while PK analysis of tail vein blood spots confirms drug-drug interactions. Fig S7 Dose-finding therapeutic study defines a once daily treatment schedule of AZD6244, 10mg/kg p.o., and AZD8055, 20mg/kg p.o., as well tolerated and active when given in combination

Supplementary Methods - PDF file 44K, Method for the preparation and analysis of AZD8055, AZD6244 and BEZ235 by LC-MS/MS Drug-drug interaction assay

Purpose: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths.Experimental Design: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA–mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations.Results: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP–ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug–drug interaction with both combinations.Conclusions: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor. Clin Cancer Res; 19(21); 5940–51. ©2013 AACR.

Supplementary Table and Figures - PDF file 184K, Suppl Table S1: Immunohistochemical analysis indicates correlation between pAKT(Ser473),pERK1/2(Thr202/Tyr204) and pS6(Ser235/236) in primary tumours Fig S1. High level expression of p110delta in two of four ARMS cell lines. Fig S2. PIK3CA KD does not induce cell cycle arrest or apoptosis in the growth inhibited RMS cells. Fig S3. Downregulation of PTEN expression appears to be associated with delayed inhibition of AKT phosphorylation in RH30 cells following p110� KD. Fig S4 Increased sensitivity of the RMS-1 PIK3CA KD line to TGX221, and of the RMS-1, RD and RMS-YM KD lines to ZSTK474. Fig S5 Equivalent changes in the biomarkers of MAPK and PI3K activity at 6h in vivo, to those seen in vitro following acute dosing with AZD6244 and AZD8055 alone, and in combination, with recovery to control levels correlating with plasma and tumor clearance of both drugs. Fig S6 Tolerability study of twice daily dosing of 10mk/kg po AZD6244 and AZD8055 indicates reversible weight loss with combination treatment, while PK analysis of tail vein blood spots confirms drug-drug interactions. Fig S7 Dose-finding therapeutic study defines a once daily treatment schedule of AZD6244, 10mg/kg p.o., and AZD8055, 20mg/kg p.o., as well tolerated and active when given in combination

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein–protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.