In 1982, the RAS genes HRAS and KRAS were discovered as the first human cancer genes, with KRAS later identified as one of the most frequently mutated oncogenes. Yet, it took nearly 40 years to develop clinically effective inhibitors for RAS-mutant cancers. The discovery in 2013 by Shokat and colleagues of a druggable pocket in KRAS paved the way to FDA approval of the first covalently binding KRAS^G12C inhibitors, sotorasib and adagrasib, in 2021 and 2022, respectively. However, rather than marking the end of a successful assault on the Mount Everest of cancer research, this landmark only revealed new challenges in RAS drug discovery. In this review, we highlight the progress on defining resistance mechanisms and developing combination treatment strategies to improve patient responses to KRAS therapies.

The vast majority of human tumors with p53 mutations undergo loss of the remaining wildtype p53 allele (loss-of-heterozygosity, p53LOH). p53LOH has watershed significance in promoting tumor progression. However, driving forces for p53LOH are poorly understood. Here we identify the repressive WTp53–HSF1 axis as one driver of p53LOH. We find that the WTp53 allele in AOM/DSS chemically-induced colorectal tumors (CRC) of p53^R248Q/+ mice retains partial activity and represses heat-shock factor 1 (HSF1), the master regulator of the proteotoxic stress response (HSR) that is ubiquitously activated in cancer. HSR is critical for stabilizing oncogenic proteins including mutp53. WTp53-retaining CRC tumors, tumor-derived organoids and human CRC cells all suppress the tumor-promoting HSF1 program. Mechanistically, retained WTp53 activates CDKN1A/p21, causing cell cycle inhibition and suppression of E2F target MLK3. MLK3 links cell cycle with the MAPK stress pathway to activate the HSR response. In p53^R248Q/+ tumors WTp53 activation by constitutive stress represses MLK3, thereby weakening the MAPK-HSF1 response necessary for tumor survival. This creates selection pressure for p53LOH which eliminates the repressive WTp53-MAPK-HSF1 axis and unleashes tumor-promoting HSF1 functions, inducing mutp53 stabilization enabling invasion.

Cancer chemotherapy relies on a high ratio of toxicity toward cancer cells vs. nonmalignant cells, making it desirable to protect normal cells. Among the nonmalignant cells, epithelia of the gut belong to the most vulnerable ones toward chemotherapeutics. Here, we use a murine intestinal organoid model to assess a strategy for protecting such epithelia against chemotherapy. Cell cycle progression was first stalled by palbociclib, a clinically established cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Washout of the drug allowed subsequent outgrowth of gut organoids. This transient cell cycle arrest conferred near-complete protection of the cells toward the nucleoside analogue gemcitabine. Moreover, pre-treatment with palbociclib protected the organoids against SN-38, the topoisomerase I-inhibiting metabolite of irinotecan, which is otherwise known for its severe gastrointestinal toxicities. In contrast, RB1-mutated cancer cells were not protected against gemcitabine or SN-38 when pre-treated with palbociclib. Taken together, these results outline a strategy for protecting nonmalignant cells against the toxicities of chemotherapeutics commonly used to treat advanced colorectal and pancreatic cancer. We propose that this strategy is particularly promising to protect the gut when treating RB1-deficient tumors that fail to arrest the cell cycle in response to CDK4/6 inhibitors. [Figure: see text].

Resistance limits the clinical efficacy of RAS inhibitors. We applied chemical and genetic screens and identified the AXL receptor tyrosine kinase as a driver of resistance to RAS-ERK inhibition. We determined that combination treatment with the AXL inhibitor bemcentinib (AXLi) together with the RAS(ON) multi-selective tri-complex inhibitor RMC-7977 (RASi) or the ERK-selective inhibitor SCH772984 (ERKi) significantly enhanced growth suppression in human KRAS -mutant pancreatic and lung cancer models. Combined AXLi and RASi treatment of human KRAS -mutant pancreatic cell line−derived xenograft tumors synergistically suppressed ERK activation and MYC expression, and caused tumor regression. Analyses of immunocompetent mouse allograft pancreatic tumor models revealed a largely tumor cell−intrinsic response to inhibitor treatment. We identified an unexpected mechanism whereby KRAS inhibition upregulated the AXL ligand GAS6, activating AXL but inducing an AXL-dependent adaptive resistance mechanism wherein AXL antagonizes RASi efficacy. Our observations support concurrent AXL inhibition as a strategy to enhance RAS inhibitor clinical efficacy. STATEMENT OF SIGNIFICANCE: Our findings identify AXL as a driver of resistance to RAS inhibitors, establishing a combination strategy to overcome resistance and enhance RAS inhibitor therapeutic efficacy in KRAS -mutant cancer by maximally inhibiting oncogenic RAS signaling.

The stress-associated molecular chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing hundreds of oncoproteins and disturbing the stoichiometry of protein complexes. Most inhibitors target the key component heat-shock protein 90 (HSP90). However, although classical HSP90 inhibitors are highly tumor-selective, they fail in phase 3 clinical oncology trials. These failures are at least partly due to an interference with a negative feedback loop by HSP90 inhibition, known as heat-shock response (HSR): in response to HSP90 inhibition there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock factor 1 (HSF1). We recently identified that wildtype p53 (p53) actively reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here we test the hypothesis that in HSP90-based therapies simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors. Indeed, we find that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids and patient-derived organoids (PDOs). Mechanistically, upon combination therapy human CRC cells strongly upregulate p53-associated pathways, apoptosis, and inflammatory immune pathways. Likewise, in the chemical AOM/DSS CRC model in mice, dual HSF1-HSP90 inhibition strongly represses tumor growth and remodels immune cell composition, yet displays only minor toxicities in mice and normal mucosa-derived organoids. Importantly, inhibition of the cyclin dependent kinases 4 and 6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Even more important, in p53-deficient (mutp53-harboring) CRC cells, an HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR system and reduces cancer growth. Likewise, p53-mutated PDOs strongly respond to dual HSF1-HSP90 pathway inhibition and thus, providing a strategy to target CRC independent of the p53 status. In sum, activating p53 (in p53-proficient cancer cells) or inhibiting CDK4/6 (independent of the p53 status) provide new options to improve the clinical outcome of HSP90-based therapies and to enhance colorectal cancer therapy.

A prerequisite for gain-of-function (GOF) p53 missense mutants (mutp53) is protein stabilization. Moreover, a prerequisite for mutp53 stabilization is loss of the remaining wildtype (WT) p53 allele (loss-of-heterozygosity, p53LOH) in mutp53/+ tumors. Thus, GOF, mutp53 stabilization and p53LOH are strictly linked. However, the driving force for p53LOH is unknown. Typically, heterozygous tumors are an instable transition state. Here we identify the repressive WTp53-HSF1 axis as the driver of p53LOH. We find that the WTp53 allele in AOM/DSS-induced colorectal tumors (CRC) of p53 ^R248Q/+ mice retains its haploid transcriptional activity. Notably, WTp53 represses heat-shock factor 1 (HSF1) activity, the master transcription factor of the proteotoxic stress defense response (HSR) that is ubiquitously and constitutively activated in cancer tissues. HSR is critical for stabilizing oncogenic proteins including mutp53. WTp53-retaining murine CRC tumors and tumor-derived organoids and human CRC cells all suppress the tumor-promoting HSF1 transcriptional program. Mechanistically, the retained WTp53 allele activates CDKN1A/p21, leading to cell cycle inhibition and suppression of the E2F target gene MLK3. MLK3 links cell cycle to the MAPK stress pathway to activate the HSR response. We show that in p53 ^R248Q/+ tumors WTp53 activation by constitutive stress (emanating from proliferative/metabolic stresses and genomic instability) represses MLK3, consequently inactivating the MAPK-HSF1 response necessary to ensure tumor survival. This creates strong selection pressure for p53LOH which eliminates the repressive WTp53-HSF1 axis and unleashes the tumor-promoting HSF1 functions, inducing mutp53 stabilization and enabling invasion. HIGHLIGHTS: heterozygous p53 ^R248Q/+ tumors retain p53 transcriptional activity in a mouse model of colorectal cancer (CRC) wildtype p53 actively represses the tumor-promoting HSF1-regulated chaperone system and proteotoxic stress response the repressive WTp53 – HSF1 axis creates a selective pressure for WTp53 loss-of-heterozygosity in CRC tumors p53 loss-of-heterozygosity enables stabilization of the gain-of-function p53 ^R248Q mutant protein which in turn enables CRC invasion