Targeting KRAS in cancer.

RAS family variants-most of which involve KRAS-are the most commonly occurring hotspot mutations in human cancers and are associated with a poor prognosis. For almost four decades, KRAS has been considered undruggable, in part due to its structure, which lacks small-molecule binding sites. But recent developments in bioengineering, organic chemistry and related fields have provided the infrastructure to make direct KRAS targeting possible. The first successes occurred with allele-specific targeting of KRAS p.Gly12Cys (G12C) in non-small cell lung cancer, resulting in regulatory approval of two agents-sotorasib and adagrasib. Inhibitors targeting other variants beyond G12C have shown preliminary antitumor activity in highly refractory malignancies such as pancreatic cancer. Herein, we outline RAS pathobiology with a focus on KRAS, illustrate therapeutic approaches across a variety of malignancies, including emphasis on the 'on' and 'off' switch allele-specific and 'pan' RAS inhibitors, and review immunotherapeutic and other key combination RAS targeting strategies. We summarize mechanistic understanding of de novo and acquired resistance, review combination approaches, emerging technologies and drug development paradigms and outline a blueprint for the future of KRAS therapeutics with anticipated profound clinical impact.

Alveolar Differentiation Drives Resistance to KRAS Inhibition in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD), commonly driven by KRAS mutations, is responsible for 7% of all cancer mortality. The first allele-specific KRAS inhibitors were recently approved in LUAD, but the clinical benefit is limited by intrinsic and acquired resistance. LUAD predominantly arises from alveolar type 2 (AT2) cells, which function as facultative alveolar stem cells by self-renewing and replacing alveolar type 1 (AT1) cells. Using genetically engineered mouse models, patient-derived xenografts, and patient samples, we found inhibition of KRAS promotes transition to a quiescent AT1-like cancer cell state in LUAD tumors. Similarly, suppressing Kras induced AT1 differentiation of wild-type AT2 cells upon lung injury. The AT1-like LUAD cells exhibited high growth and differentiation potential upon treatment cessation, whereas ablation of the AT1-like cells robustly improved treatment response to KRAS inhibitors. Our results uncover an unexpected role for KRAS in promoting intratumoral heterogeneity and suggest that targeting alveolar differentiation may augment KRAS-targeted therapies in LUAD. SIGNIFICANCE: Treatment resistance limits response to KRAS inhibitors in LUAD patients. We find LUAD residual disease following KRAS targeting is composed of AT1-like cancer cells with the capacity to reignite tumorigenesis. Targeting the AT1-like cells augments responses to KRAS inhibition, elucidating a therapeutic strategy to overcome resistance to KRAS-targeted therapy. This article is featured in Selected Articles from This Issue, p. 201.

A classical epithelial state drives acute resistance to KRAS inhibition in pancreas cancer.

Intra-tumoral heterogeneity in pancreatic ductal adenocarcinoma (PDAC) is characterized by a balance between basal and classical epithelial cancer cell states, with basal dominance associating with chemoresistance and a dismal prognosis. Targeting oncogenic KRAS, the primary driver of pancreatic cancer, shows early promise in clinical trials but efficacy is limited by acquired resistance. Using genetically engineered mouse models and patient-derived xenografts, we find that basal PDAC cells are highly sensitive to KRAS inhibitors. Employing fluorescent and bioluminescent reporter systems, we longitudinally track cell-state dynamics in vivo and reveal a rapid, KRAS inhibitor-induced enrichment of the classical state. Lineage-tracing identifies these enriched classical PDAC cells to be a reservoir for disease relapse. Genetic ablation of the classical cell-state is synergistic with KRAS inhibition, providing a pre-clinical proof-of-concept for this therapeutic strategy. Our findings motivate combining classical-state directed therapies with KRAS inhibitors to deepen responses and counteract resistance in pancreatic cancer.

Precision medicine for pancreatic cancer: characterizing the clinicogenomic landscape and outcomes of KRAS G12C-mutated disease.

BACKGROUND: Mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogene alteration in pancreatic ductal adenocarcinoma, and KRAS glycine to cystine substitution at codon 12 (G12C) mutations (KRAS G12Cmut) are observed in 1%-2%. Several inhibitors of KRAS G12C have recently demonstrated promise in solid tumors, including pancreatic cancer. Little is known regarding clinical, genomics, and outcome data of this population. METHODS: Patients with pancreatic cancer and KRAS G12Cmut were identified at Memorial Sloan Kettering Cancer Center and via the American Association of Cancer Research Project Genomics, Evidence, Neoplasia, Information, Exchange database. Clinical, treatment, genomic, and outcomes data were analyzed. A cohort of patients at Memorial Sloan Kettering Cancer Center with non-G12C KRAS pancreatic cancer was included for comparison. RESULTS: Among 3571 patients with pancreatic ductal adenocarcinoma, 39 (1.1%) with KRAS G12Cmut were identified. Median age was 67 years, and 56% were female. Median body mass index was 29.2 kg/m2, and 67% had a smoking history. Median overall survival was 13 months (95% CI: 9.4 months, not reached) for stage IV and 26 months (95% CI: 23 months, not reached) for stage I-III. Complete genomic data (via American Association of Cancer Research Project Genomics, Evidence, Neoplasia, Information, Exchange database) was available for 74 patients. Most common co-alterations included TP53 (73%), CDKN2A (33%), SMAD4 (28%), and ARID1A (21%). Compared with a large cohort (n = 2931) of non-G12C KRAS-mutated pancreatic ductal adenocarcinoma, ARID1A co-mutations were more frequent in KRAS G12Cmut (P < .05). Overall survival did not differ between KRAS G12Cmut and non-G12C KRAS pancreatic ductal adenocarcinoma. Germline pathogenic variants were identified in 17% of patients; 2 patients received KRAS G12C-directed therapy. CONCLUSION: Pancreatic cancer and KRAS G12Cmut may be associated with a distinct clinical phenotype. Genomic features are similar to non-G12C KRAS-mutated pancreatic cancer, although enrichment of ARID1A co-mutations was observed. Targeting of KRAS G12C in pancreatic cancer provides a precedent for broader KRAS targeting in pancreatic cancer.

Alveolar differentiation drives resistance to KRAS inhibition in lung adenocarcinoma.

Lung adenocarcinoma (LUAD), commonly driven by KRAS mutations, is responsible for 7% of all cancer mortality. The first allele-specific KRAS inhibitors were recently approved in LUAD, but clinical benefit is limited by intrinsic and acquired resistance. LUAD predominantly arises from alveolar type 2 (AT2) cells, which function as facultative alveolar stem cells by self-renewing and replacing alveolar type 1 (AT1) cells. Using genetically engineered mouse models, patient-derived xenografts, and patient samples we found inhibition of KRAS promotes transition to a quiescent AT1-like cancer cell state in LUAD tumors. Similarly, suppressing Kras induced AT1 differentiation of wild-type AT2 cells upon lung injury. The AT1-like LUAD cells exhibited high growth and differentiation potential upon treatment cessation, whereas ablation of the AT1-like cells robustly improved treatment response to KRAS inhibitors. Our results uncover an unexpected role for KRAS in promoting intra-tumoral heterogeneity and suggest targeting alveolar differentiation may augment KRAS-targeted therapies in LUAD. Significance: Treatment resistance limits response to KRAS inhibitors in LUAD patients. We find LUAD residual disease following KRAS targeting is composed of AT1-like cancer cells with the capacity to reignite tumorigenesis. Targeting the AT1-like cells augments responses to KRAS inhibition, elucidating a therapeutic strategy to overcome resistance to KRAS-targeted therapy.

Solid tumor growth depends on an intricate equilibrium of malignant cell states.

Control of cell identity and number is central to tissue function, yet principles governing organization of malignant cells in tumor tissues remain poorly understood. Using mathematical modeling and candidate-based analysis, we discover primary and metastatic pancreatic ductal adenocarcinoma (PDAC) organize in a stereotypic pattern whereby PDAC cells responding to WNT signals (WNT-R) neighbor WNT-secreting cancer cells (WNT-S). Leveraging lineage-tracing, we reveal the WNT-R state is transient and gives rise to the WNT-S state that is highly stable and committed to organizing malignant tissue. We further show that a subset of WNT-S cells expressing the Notch ligand DLL1 form a functional niche for WNT-R cells. Genetic inactivation of WNT secretion or Notch pathway components, or cytoablation of the WNT-S state disrupts PDAC tissue organization, suppressing tumor growth and metastasis. This work indicates PDAC growth depends on an intricately controlled equilibrium of functionally distinct cancer cell states, uncovering a fundamental principle governing solid tumor growth and revealing new opportunities for therapeutic intervention.

Systematic Comparison of Pancreatic Ductal Adenocarcinoma Models Identifies a Conserved Highly Plastic Basal Cell State.

Intratumoral heterogeneity and cellular plasticity have emerged as hallmarks of cancer, including pancreatic ductal adenocarcinoma (PDAC). As PDAC portends a dire prognosis, a better understanding of the mechanisms underpinning cellular diversity in PDAC is crucial. Here, we investigated the cellular heterogeneity of PDAC cancer cells across a range of in vitro and in vivo growth conditions using single-cell genomics. Heterogeneity contracted significantly in two-dimensional and three-dimensional cell culture models but was restored upon orthotopic transplantation. Orthotopic transplants reproducibly acquired cell states identified in autochthonous PDAC tumors, including a basal state exhibiting coexpression and coaccessibility of epithelial and mesenchymal genes. Lineage tracing combined with single-cell transcriptomics revealed that basal cells display high plasticity in situ. This work defines the impact of cellular growth conditions on phenotypic diversity and uncovers a highly plastic cell state with the capacity to facilitate state transitions and promote intratumoral heterogeneity in PDAC. SIGNIFICANCE: This work provides important insights into how different model systems of pancreatic ductal adenocarcinoma mold the phenotypic space of cancer cells, highlighting the power of in vivo models.

Glutamate spillover in C. elegans triggers repetitive behavior through presynaptic activation of MGL-2/mGluR5.

Glutamate is a major excitatory neurotransmitter, and impaired glutamate clearance following synaptic release promotes spillover, inducing extra-synaptic signaling. The effects of glutamate spillover on animal behavior and its neural correlates are poorly understood. We developed a glutamate spillover model in Caenorhabditis elegans by inactivating the conserved glial glutamate transporter GLT-1. GLT-1 loss drives aberrant repetitive locomotory reversal behavior through uncontrolled oscillatory release of glutamate onto AVA, a major interneuron governing reversals. Repetitive glutamate release and reversal behavior require the glutamate receptor MGL-2/mGluR5, expressed in RIM and other interneurons presynaptic to AVA. mgl-2 loss blocks oscillations and repetitive behavior; while RIM activation is sufficient to induce repetitive reversals in glt-1 mutants. Repetitive AVA firing and reversals require EGL-30/Gαq, an mGluR5 effector. Our studies reveal that cyclic autocrine presynaptic activation drives repetitive reversals following glutamate spillover. That mammalian GLT1 and mGluR5 are implicated in pathological motor repetition suggests a common mechanism controlling repetitive behaviors.

EFF-1 fusogen promotes phagosome sealing during cell process clearance in Caenorhabditis elegans.

Phagocytosis of dying cells is critical in development and immunity1-3. Although proteins for recognition and engulfment of cellular debris following cell death are known4,5, proteins that directly mediate phagosome sealing are uncharacterized. Furthermore, whether all phagocytic targets are cleared using the same machinery is unclear. Degeneration of morphologically complex cells, such as neurons, glia and melanocytes, produces phagocytic targets of various shapes and sizes located in different microenvironments6,7. Thus, such cells offer unique settings to explore engulfment programme mechanisms and specificity. Here, we report that dismantling and clearance of a morphologically complex Caenorhabditis elegans epithelial cell requires separate cell soma, proximal and distal process programmes. Similar compartment-specific events govern the elimination of a C. elegans neuron. Although canonical engulfment proteins drive cell soma clearance, these are not required for process removal. We find that EFF-1, a protein previously implicated in cell-cell fusion 8 , specifically promotes distal process phagocytosis. EFF-1 localizes to phagocyte pseudopod tips and acts exoplasmically to drive phagosome sealing. eff-1 mutations result in phagocytosis arrest with unsealed phagosomes. Our studies suggest universal mechanisms for dismantling morphologically complex cells and uncover a phagosome-sealing component that promotes cell process clearance.

Infrared laser-induced gene expression for tracking development and function of single C. elegans embryonic neurons.

Visualizing neural-circuit assembly in vivo requires tracking growth of optically resolvable neurites. The Caenorhabditis elegans embryonic nervous system, comprising 222 neurons and 56 glia, is attractive for comprehensive studies of development; however, embryonic reporters are broadly expressed, making single-neurite tracking/manipulation challenging. We present a method, using an infrared laser, for reproducible heat-dependent gene expression in small sublineages (one to four cells) without radiation damage. We go beyond proof-of-principle, and use our system to label and track single neurons during early nervous-system assembly. We uncover a retrograde extension mechanism for axon growth, and reveal the aetiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants. We also perform cell-specific rescues, determining DAF-6/patched-related site of action during sensory-organ development. Simultaneous ablation and labelling of cells using our system reveals roles for glia in dendrite extension. Our method can be applied to other optically/IR-transparent organisms, and opens the door to high-resolution systematic analyses of C. elegans morphogenesis.