Preclinical efficacy of carfilzomib in BRAF-mutant colorectal cancer models.

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T-cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients.

Temozolomide Treatment Alters Mismatch Repair and Boosts Mutational Burden in Tumor and Blood of Colorectal Cancer Patients.

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, whereas MMR-deficient (MMRd) tumors often respond to immune-checkpoint blockade. We previously reported that the treatment of colorectal cancer preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB), and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-methylguanine-DNA-methyltransferase (MGMT)-deficient, MMR-proficient, RAS-mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A subset of patients whose tumors displayed the MSH6 mutation, the TMZ mutational signature, and increased TMB achieved disease stabilization upon pembrolizumab treatment. SIGNIFICANCE: MMR-proficient mCRCs are unresponsive to immunotherapy. We provide the proof of concept that inactivation of MMR genes can be achieved pharmacologically with TMZ and molecularly monitored in the tissue and blood of patients with mCRC. This strategy deserves additional evaluation in mCRC patients whose tumors are no longer responsive to standard-of-care treatments. See related commentary by Willis and Overman, p. 1612. This article is highlighted in the In This Issue feature, p. 1599.