POSTER ESMO GI 2025 124P Final.pdf

G12X

Synthetic Lethality of CBM Signalosome Inhibition for KRAS Colorectal Cancer

Authors

Yi Yao, Kevin Ling, Huili Xia, Yiling Qi, Huadong Sun, Chunxuan Shao, Sharon Wu, Wei Liu, Gerry Harriman, Fang Wang
HotSpot Therapeutics, Inc, One Design Center Place, Suite 19-600, Boston, MA 02210, USA; Caris Life Science, 750 W John Carpenter Fwy, Suite 800, Irving, TX 75039, USA

Introduction

The CBM signalosome (CARD11-BCL10-MALT1) is a signaling hub directly regulating multiple oncogenic pathways, including NFκB, JNK, mTORC1 and MYC. This positions the CBM complex as a critical regulator of tumor development and survival. For the first time, we uncovered the essential pro-survival role of the CBM signalosome in G12 KRAS-driven colorectal cancers (CRCs). This discovery may provide a therapeutic breakthrough for difficult-to-treat KRAS-driven CRCs.

CBM Signalosome, the Pro-Survival Hub for Cancer Progression

Results

Scaffolding assay: OCI-LY3 cells were treated with HOT-103 for 24hr, the modulation of p-IκBα was monitored in MSD format.
NFkB assay: Jurkat cells were pre-treated with HOT-103 for 1 hr, followed by stimulation with anti-CD3/anti-CD28/PMA for 4 hr, and NFkB reporter activity was measured using a luciferase assay.
AP1 assay: HEK293-AP1 cells were pre-treated with HOT-103 for 1 hr, followed by stimulation with 10 nM PMA for 6 hr, and AP1 reporter activity was measured using a luciferase assay.
mTORC1 assay: OCI-LY3 were treated with HOT-103 (0.3, 1, 3 M), and rapamycin (10 nM) for 24hrs. The phosphorylated ribosomal protein S6 (Ser240) was analyzed by flowcytometry.

CBM transduces upstream growth & survival signals via a series of phosphorylation and ubiquitination reactions, and activates multiple pro-survival pathways, including canonical NFκB, mTORC1 and JNK. In cancer, upstream GoFs (BTK, PKC,

mTORC1 Activity Assay

CBMi Induced Profound Apoptosis in KRAS CRC Cells

A panel of CRC cell lines were treated with CBM inhibitor (HOT-051) or BCL2/Bcl-xL inhibitor (Navitoclax) in dose and time-dependent manner. The plot was graphed based on 3 M of compounds (maximum inhibition) after 96hr treatment. 0-100: growth inhibition; 0: growth stasis; <0%: cell death. Depmap CRISPR knockout screen discovered KRAS-active CRC showed modest sensitivity to Bcl-xL knockdown. In head-to-head comparison, CBMi was superior to Navitoclax. Venetoclax had no effect on CRC lines (data not shown).

CBMi HOT-051

Pan-KRASi: RMC-6236

CBMi Plus KRASi Achieved Durable MAPK Suppression

Higher CARD11 is significantly associated with high KRAS/NFκB/Apoptosis activities in pan-cancer studies, specifically in high KRASm prevalence cancer indications.

(A) HEK293 cells were transiently transduced with KRAS or KRAS. After 24hr of treatment with 1 μM HOT-051 or RMC-6236, cells were lysed and protein levels were analyzed by Western blot.
(B) SW620 (KRAS) cells were treated with HOT-051 or RMC-6236 for 24hr, followed by Western blot analysis of protein expression. (C) SW837 cells (KRAS) were treated with HOT-103 (CBMi), RMC-6236, or the combination for 1hr or 24hr. Cells were then lysed, and protein expression was assessed by Western blot. Similar results were also observed in multiple KRAS cell lines.

Mice studies

Mice were treated orally with HOT-051, Adagrasib, or the combination at varying doses for 18 days. (A) Tumor volume at the end of study, (B) changes of mouse body weight, and (C) individual tumor growth percentages are shown.