# 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.

- KRAS tumor hijacked CBM signalosome for survival.

- To our knowledge, CBMi is the first molecule that selectively induced apoptosis in KRAS tumor and blocked tumor growth in in vitro and in vivo preclinical models.