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MSS/KRAS CRC Depends on CBM Signalosome for Survival - G12X

A Discovery that May Transform KRAS CRC Therapy

Introduction

KRAS activation is a prominent genetic feature of colorectal cancer (CRC), however, CRC patients did not demonstrate deep or durable responses when treated with KRAS inhibitors.

We and others have observed that KRAS inhibitors, while potently inhibiting cancer proliferation, do not induce strong apoptosis in vitro, in vivo or in patients with CRC. This has led to limited and non-durable clinical response of KRASi, because tumor cells remain alive and soon relapse or develop resistance.

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We discovered that MSS/KRAS CRC depends on CBM signalosome for survival. Inhibition of CBM selectively induces selective and potent apoptosis in KRAS CRC.

CBM Signalosome, the Pro-Survival Hub for Cancer Progression

RTK, GPCR, TCR, BCR

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 vitro data was from Revolution Medicine and HotSpot’s data; the rate represents the percentage of cell lines responding to KRASi induced anti-proliferation or apoptosis in a cell panel screen.

Lack of Potent Apoptosis Could Explain the Limited and Non-Durable Response of KRASi in CRC

Results

Figure 1. In CRC, High CARD11 CRC had Highest KRAS Activity and Shorter KRASi Time-on-Treatment

CARD11 CNA or Amp were Discovered in >50% of MSS CRC Patients

A Study of > 20,000 CRC Patients Database Revealed CARD11 CNA or Amp in >50% of MSS CRC

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CARD11 CRC had Shorter Time-on-Treatment of KRAS Inhibitor Adagrasib/Sotorasib Time on Treatment (TOT)

Same Database Revealed High CARD11 CRC has the Highest KRAS Activity Among Oncogenic Pathways (P< 0.0001)

# patients who received Sotorasib or Adgrasib in the group of interest

• Q1: Lowest 25% CARD11 expression

• N=50 treated with Sotorasib (N=46 treated with only Sotorasib) • N=35 treated with Adagrasib (N=31 treated with only Adagrasib) • N=4 treated with both Sotorasib and Adagrasib

# patients who received Sotorasib or Adgrasib in the group of interest

• Q1: Lowest 25% CARD11 expression ** Q4: Highest 25% CARD11 expression

Figure 2. CBM Inhibitor Selectively Induced Apoptosis in G12X MSS/KRAS CRC

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CBM Inhibitor Selectively Induced Apoptosis in MSS/KRAS CRC

Part 1: Tumor Regression After 7-day Treatment

Cells were treated with HOT-103 (CBMi) for 24hr, followed by Western blot analysis of protein expression.

(A) A panel of CRC lines (including KRAS WT and KRAS mutant) were treated with CBM inhibitor (HOT-051) in dose and time-dependent manner. The plot was graphed based on 3 M of HOT-051 (maximum inhibition) after 96hr treatment. 0-100: growth inhibition; 0: growth stasis; <-20%: cell death. (B) Representative cell lines for HOT-051 induced apoptosis. Dose and time-dependent caspase 3/7 cleavage was monitored using Incucyte.

Figure 3. CBM Inhibitor Induced Potent Apoptosis in G12X MSS/KRAS CRC, Superior to KRASi Plus EGFR Antibody

Resistant CRC

Figure 4. CBM Inhibitors Blocked Multiple Oncogenic Survival G12X Signals in Sensitive KRAS CRC

Conclusions

• KRAS inhibitors do not induce strong apoptosis in vitro, in vivo, or in patients, especially for CRC, resulting in limited and non-durable response of KRASi.

• We discovered that KRAS CRC depends on the CBM signalosome for survival. Inhibition of CBM signalosome induced profound apoptosis/cell death of KRAS CRC in in vitro and in vivo preclinical models.