Abstract 1735: Identification of novel synthetic lethal interactions in small cell lung cancer with the proteasome inhibitor carfilzomib
Authors: Masto Vincent B. , Lerner Alana G. , Arastu-Kapur Shirin 
Published: August 2015

Abstract
Proteasome inhibition (PI) has emerged as a valuable cancer treatment in hematological malignancies by causing a disruption of protein homeostasis. While proteasome inhibitors have been historically unsuccessful in the clinic for solid tumors, all cancer cell lines succumb to proteasome inhibition in vitro. Solid tumor cell lines have a higher threshold for disruption of protein homeostasis; as they require continuous exposure to achieve cytotoxicity similar to pulsatile exposure of PI in heme cell lines. The in-vivo half-life of proteasome inhibitors is short (∼1hr) making it challenging to mimic in vitro continuous treatment. Therefore we explored the option of sensitizing solid tumors to proteasome inhibitors through combination therapy.
In order to identify novel synthetic lethal interactions with PI, we performed a screen utilizing a pooled siRNA library targeting 2240 genes of the druggable genome in combination with the proteasome inhibitor carfilzomib (CFZ) or DMSO. Cells were first transfected with the siRNA for 24 hours and then treated with CFZ or DMSO for an additional 72h to assess synergy. We performed the screen in small cell lung cancer (SCLC), an unmet medical need. Preliminary responses in SCLC patients were observed with CFZ in an all comers solid tumor trial (Papadopoulos, et al. Cancer Chemother Pharmacol, 2013).
The screen was performed in NCI-H841 cells that are adherent, easy to culture, and have high transfection efficiency. The combined effect of siRNA and CFZ on viability was measured using a Sensitivity Index (SI), which ranges from -1 (antagonistic) to 1 (synergistic). Using an SI threshold &gt;0.2, we identified 33 synthetic lethal genes when H841 cells were treated with an IC9 of CFZ.
To validate screen hits, we performed experiments using single siRNA reagents (4 - 6 sequences per gene) in combination with CFZ in NCI-H841. Two hits were confirmed with two or more single siRNAs sensitizing cells to CFZ. These genes (TGFBR3 and GRK6) are of particular interest because of their involvement in proliferation signaling mechanisms. Subsequent screens have also uncovered two additional genes, involved in the protein degradation and trafficking pathway (CLPP and COG8), which meet the same validation criteria for synergy.
The characterization of these hits is currently on-going in a panel of SCLC lines, and in cells engineered with CRISPR technology to achieve a near complete knockout. In parallel, we are testing commercially available reagents targeting our proteins of interest to validate our results. Preliminary data with a published GRK6 inhibitor, GF109203x, demonstrates synergy with Bliss scores of 0.35 in H841 and 0.44 in DMS114 SCLC lines (Zhou et al. J Pharmacol Exp Ther, 2001). In summary, we have identified druggable targets that would potentiate CFZ efficacy towards solid tumors and may elucidate the PI mechanism of action.
Citation Format: Vincent B. Masto, Alana G. Lerner, Shirin Arastu-Kapur. Identification of novel synthetic lethal interactions in small cell lung cancer with the proteasome inhibitor carfilzomib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1735. doi:10.1158/1538-7445.AM2015-1735