Cytokine CCL9 Mediates Oncogenic KRAS-Induced Pancreatic Acinar-to-Ductal Metaplasia by Promoting Reactive Oxygen Species and Metalloproteinases.

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48cre:KrasG12D mice and human PDAC patients. Knockdown of CCL9 in KrasG12D-expressed pancreatic acini reduced KrasG12D-induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of KrasG12D, CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48cre:KrasG12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.

Construction and validation of "WCH-nomogram" for predicting the prognosis after resection of colorectal liver metastases.

BACKGROUND: The prognostic predictive tool for patients with colorectal liver metastasis (CRLM) is limited and the criteria for administering preoperative neoadjuvant chemotherapy in CRLM patients remain controversial. METHODS: This study enrolled 532 CRLM patients at West China Hospital (WCH) from January 2009 to December 2019. Prognostic factors were identified from the training cohort to construct a WCH-nomogram and evaluating accuracy in the validation cohort. Receiver operating characteristic (ROC) curve analysis was used to compare the prediction accuracy with other existing prediction tools. RESULTS: From the analysis of the training cohort, four independent prognostic risk factors, namely tumor marker score, KRAS mutation, primary lymph node metastasis, and tumor burden score were identified on which a WCH-nomogram was constructed. The C-index of the two cohorts were 0.674 (95% CI: 0.634-0.713) and 0.655 (95% CI: 0.586-0.723), respectively, which was better than the previously reported predication scores (CRS, m-CS and GAME score). ROC curves showed AUCs for predicting 1-, 3-, and 5-year overall survival (OS) of 0.758, 0.709, and 0.717 in the training cohort, and 0.860, 0.669, and 0.692 in the validation cohort, respectively. A cutoff value of 114.5 points was obtained for the WCH-nomogram total score based on the maximum Youden index of the ROC curve of 5-year OS. Risk stratification showed significantly better prognosis in the low-risk group, however, the high-risk group was more likely to benefit from neoadjuvant chemotherapy. CONCLUSIONS: The WCH-nomogram demonstrates superior prognostic stratification compared to prior scoring systems, effectively identifying CRLM patients who may benefit the most from neoadjuvant chemotherapy.

Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

Protein phosphatase 6 activates NF-κB to confer sensitivity to MAPK pathway inhibitors in KRAS- and BRAF-mutant cancer cells.

The Ras-mitogen-activated protein kinase (MAPK) pathway is a major target for cancer treatment. To better understand the genetic pathways that modulate cancer cell sensitivity to MAPK pathway inhibitors, we performed a CRISPR knockout screen with MAPK pathway inhibitors on a colorectal cancer (CRC) cell line carrying mutant KRAS. Genetic deletion of the catalytic subunit of protein phosphatase 6 (PP6), encoded by PPP6C, rendered KRAS- and BRAF-mutant CRC and BRAF-mutant melanoma cells more resistant to these inhibitors. In the absence of MAPK pathway inhibition, PPP6C deletion in CRC cells decreased cell proliferation in two-dimensional (2D) adherent cultures but accelerated the growth of tumor spheroids in 3D culture and tumor xenografts in vivo. PPP6C deletion enhanced the activation of nuclear factor κB (NF-κB) signaling in CRC and melanoma cells and circumvented the cell cycle arrest and decreased cyclin D1 abundance induced by MAPK pathway blockade in CRC cells. Inhibiting NF-κB activity by genetic and pharmacological means restored the sensitivity of PPP6C-deficient cells to MAPK pathway inhibition in CRC and melanoma cells in vitro and in CRC cells in vivo. Furthermore, a R264 point mutation in PPP6C conferred loss of function in CRC cells, phenocopying the enhanced NF-κB activation and resistance to MAPK pathway inhibition observed for PPP6C deletion. These findings demonstrate that PP6 constrains the growth of KRAS- and BRAF-mutant cancer cells, implicates the PP6-NF-κB axis as a modulator of MAPK pathway output, and presents a rationale for cotargeting the NF-κB pathway in PPP6C-mutant cancer cells.

Molecular Deciphering of Colorectal Cancer: Exploring Molecular Classifications and Analyzing the Interplay among Molecular Biomarkers MMR/MSI, KRAS, NRAS, BRAF and CDX2 - A Comprehensive Literature Review.

Background: Colorectal cancer (CRC) exhibits molecular and morphological diversity, involving genetic, epigenetic alterations, and disruptions in signaling pathways. This necessitates a comprehensive review synthesizing recent advancements in molecular mechanisms, established biomarkers, as well as emerging ones like CDX2 for enhanced CRC assessment. Material and Methods: This review analyzes the last decade's literature and current guidelines to study CRC's molecular intricacies. It extends the analysis beyond traditional biomarkers to include emerging ones like CDX2, examining their interaction with carcinogenic mechanisms and molecular pathways, alongside reviewing current testing methodologies. Results: A multi-biomarker strategy, incorporating both traditional and emerging biomarkers like CDX2, is crucial for optimizing CRC management. This strategy elucidates the complex interaction between biomarkers and the tumor's molecular pathways, significantly influencing prognostic evaluations, therapeutic decision-making, and paving the way for personalized medicine in CRC. Conclusions: This review proposes CDX2 as an emerging prognostic biomarker and emphasizes the necessity of thorough molecular profiling for individualized treatment strategies. By enhancing CRC treatment approaches and prognostic evaluation, this effort marks a step forward in precision oncology, leveraging an enriched understanding of tumor behavior.

KRAS Allelic Variants in Biliary Tract Cancers.

Importance: Biliary tract cancers (BTCs) contain several actionable molecular alterations, including FGFR2, IDH1, ERBB2 (formerly HER2), and KRAS. KRAS allelic variants are found in 20% to 30% of BTCs, and multiple KRAS inhibitors are currently under clinical investigation. Objectives: To describe the genomic landscape, co-sequence variations, immunophenotype, genomic ancestry, and survival outcomes of KRAS-mutated BTCs and to calculate the median overall survival (mOS) for the most common allelic variants. Design, Setting, and Participants: This retrospective, multicenter, pooled cohort study obtained clinical and next-generation sequencing data from multiple databases between January 1, 2017, and December 31, 2022. These databases included Princess Margaret Cancer Centre, MD Anderson Cancer Center, Foundation Medicine, American Association for Cancer Research Project GENIE, and cBioPortal for Cancer Genomics. The cohort comprised patients with BTCs who underwent genomic testing. Main Outcome and Measure: The main outcome was mOS, defined as date of diagnosis to date of death, which was measured in months. Results: A total of 7457 patients (n = 3773 males [50.6%]; mean [SD] age, 63 [5] years) with BTCs and genomic testing were included. Of these patients, 5813 had clinical outcome data available, in whom 1000 KRAS-mutated BTCs were identified. KRAS allelic variants were highly prevalent in perihilar cholangiocarcinoma (28.6%) and extrahepatic cholangiocarcinoma (36.1%). Thirty-six KRAS allelic variants were identified, and the prevalence rates in descending order were G12D (41%), G12V (23%), and Q61H (8%). The variant G12D had the highest mOS of 25.1 (95% CI, 22.0-33.0) months compared with 22.8 (95% CI, 19.6-31.4) months for Q61H and 17.8 (95% CI, 16.3-23.1) months for G12V variants. The majority of KRAS-mutated BTCs (98.9%) were not microsatellite instability-high and had low tumor mutational burden (ranging from a median [IQR] of 1.2 (1.2-2.5) to a mean [SD] of 3.3 [1.3]). Immune profiling through RNA sequencing of KRAS and NRAS-mutated samples showed a pattern toward a more immune-inflamed microenvironment with higher M1 macrophage activation (0.16 vs 0.12; P = .047) and interferon-γ expression compared with wild-type tumors. The G12D variant remained the most common KRAS allelic variant in all patient ancestries. Patients with admixed American ancestry had the highest proportion of G12D variant (45.0%). Conclusions and Relevance: This cohort study found that KRAS allelic variants were relatively common and may be potentially actionable genomic alterations in patients with BTCs, especially perihilar cholangiocarcinoma and extrahepatic cholangiocarcinoma. The findings add to the growing data on genomic and immune landscapes of KRAS allelic variants in BTCs and are potentially of value to the planning of specific therapies for this heterogeneous patient group.

RAS G12C Inhibitors: Three Birds with One Stone.

SUMMARY: In this issue, Rubinson, Tanaka, and colleagues demonstrate that differences among G12C inhibitors rely on their ability to covalently bind not only G12C mutant KRAS but also NRAS and HRAS, proposing sotorasib as a potent NRAS G12C inhibitor. See related article by Rubinson et al., p. 727 (6).

Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Exploiting the ferroaddiction of pancreatic cancer cells using Fe-doped nanoparticles.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor survival rates. Here, we evaluated iron-doped hydroxyapatite (FeHA) as a potential nanomedicine-based approach to combat PDAC. FeHA, in combination with a sublethal dose of the glutathione peroxidase 4 (GPX4) inhibitor RSL3, was found to trigger ferroptosis in KRAS mutant PANC-1 cells, but not in BxPC3 cells, while sparing normal human cells (fibroblasts and peripheral blood mononuclear cells). These findings were recapitulated in 3D spheroids generated using PDAC cells harboring wild-type versus mutant KRAS. Moreover, ferroptosis induction by FeHA plus RSL3 was reversed by the knockdown of STEAP3, a metalloreductase responsible for converting Fe3+ to Fe2+. Taken together, our data show that FeHA is capable of triggering cancer cell death in a KRAS-selective, STEAP3-dependent manner in PDAC cells.

Quercetin Exhibits Preferential Binding Interaction by Selectively Targeting HRAS1 I-Motif DNA-Forming Promoter Sequences.

I-Motif (iM) DNA structures represent among the most significant noncanonical nucleic acid configurations. iM-forming DNA sequences are found in an array of vital genomic locations and are particularly frequent in the promoter islands of various oncogenes. Thus, iM DNA is a crucial candidate for anticancer medicines; therefore, binding interactions between iM DNA and small molecular ligands, such as flavonoids, are critically important. Extensive sets of spectroscopic strategies and thermodynamic analysis were utilized in the present investigation to find out the favorable interaction of quercetin (Que), a dietary flavonoid that has various health-promoting characteristics, including anticancer properties, with noncanonical iM DNA structure. Spectroscopic studies and thermal analysis revealed that Que interacts preferentially with HRAS1 iM DNA compared with VEGF, BCL2 iM, and duplex DNA. Que, therefore, emerged as a suitable natural-product-oriented antagonist for targeting HRAS1 iM DNA. The innovative spectroscopic as well as mechanical features of Que and its specific affinity for HRAS1 iM may be useful for therapeutic applications and provide crucial insights for the design of compounds with remarkable medicinal properties.

Inhibition of PRMT5-mediated regulation of DKK1 sensitizes colorectal cancer cells to chemotherapy.

The Dickkopf family proteins (DKKs) are strong Wnt signaling antagonists that play a significant role in colorectal cancer (CRC) development and progression. Recent work has shown that DKKs, mainly DKK1, are associated with the induction of chemoresistance in CRC and that DKK1 expression in cancer cells correlates with that of protein arginine N-methyltransferase 5 (PRMT5). This points to the presence of a regulatory loop between DKK1 and PRMT5. Herein, we addressed the question of whether PRMT5 contributes to DKK1 expression in CRC and hence CRC chemoresistance. Both in silico and in vitro approaches were used to explore the relationship between PRMT5 and different DKK members. Our data demonstrated that DKK1 expression is significantly upregulated in CRC clinical samples, KRAS-mutated CRC in particular and that the levels of DKK1 positively correlate with PRMT5 activation. Chromatin immunoprecipitation (ChIP) data indicated a possible epigenetic role of PRMT5 in regulating DKK1, possibly through the symmetric dimethylation of H3R8. Knockdown of DKK1 or treatment with the PRMT5 inhibitor CMP5 in combination with doxorubicin yielded a synergistic anti-tumor effect in KRAS mutant, but not KRAS wild-type, CRC cells. These findings suggest that PRMT5 regulates DKK1 expression in CRC and that inhibition of PRMT5 modulates DKK1 expression in such a way that reduces CRC cell growth.

A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.

The RAS oncogene in brain tumors and the involvement of let-7 microRNA.

RAS oncogenes are master regulator genes in many cancers. In general, RAS-driven cancers have an oncogenic RAS mutation that promotes disease progression (colon, lung, pancreas). In contrast, brain tumors are not necessarily RAS-driven cancers because RAS mutations are rarely observed. In particular, glioblastomas (the most lethal brain tumor) do not appear to have dominant genetic mutations that are suitable for targeted therapy. Standard treatment for most brain tumors continues to focus on maximal surgical resection, radiotherapy and chemotherapy. Yet the convergence of genomic aberrations such as EGFR, PDGFR and NF1 (some of which are clinically effective) with activation of the RAS/MAPK cascade is still considered a key point in gliomagenesis, and KRAS is undoubtedly a driving gene in gliomagenesis in mice. In cancer, microRNAs (miRNA) are small, non-coding RNAs that regulate carcinogenesis. However, the functional consequences of aberrant miRNA expression in cancer are still poorly understood. let-7 encodes an intergenic miRNA that is classified as a tumour suppressor, at least in lung cancer. Let-7 suppresses a plethora of oncogenes such as RAS, HMGA, c-Myc, cyclin-D and thus suppresses cancer development, differentiation and progression. let-7 family members are direct regulators of certain RAS family genes by binding to the sequences in their 3'untranslated region (3'UTR). let-7 miRNA is involved in the malignant behaviour in vitro-proliferation, migration and invasion-of gliomas and stem-like glioma cells as well as in vivo models of glioblastoma multiforme (GBM) via KRAS inhibition. It also increases resistance to certain chemotherapeutic agents and radiotherapy in GBM. Although let-7 therapy is not yet established, this review updates the current state of knowledge on the contribution of miRNA let-7 in interaction with KRAS to the oncogenesis of brain tumours.

Disruption of the pro-oncogenic c-RAF-PDE8A complex represents a differentiated approach to treating KRAS-c-RAF dependent PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF-PDE8A protein-protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF-cAMP/PKA signaling axes and treating KRAS-c-RAF dependent PDAC. Through disrupting the c-RAF-PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS-c-RAF dependent PDAC.

Targeted therapeutic options in early and metastatic NSCLC-overview.

The complex therapeutic strategy of non-small cell lung cancer (NSCLC) has changed significantly in recent years. Disease-free survival increased significantly with immunotherapy and chemotherapy registered in perioperative treatments, as well as adjuvant registered immunotherapy and targeted therapy (osimertinib) in case of EGFR mutation. In oncogenic-addictive metastatic NSCLC, primarily in adenocarcinoma, the range of targeted therapies is expanding, with which the expected overall survival increases significantly, measured in years. By 2021, the FDA and EMA have approved targeted agents to inhibit EGFR activating mutations, T790 M resistance mutation, BRAF V600E mutation, ALK, ROS1, NTRK and RET fusion. In 2022, the range of authorized target therapies was expanded. With therapies that inhibit KRASG12C, EGFR exon 20, HER2 and MET. Until now, there was no registered targeted therapy for the KRAS mutations, which affect 30% of adenocarcinomas. Thus, the greatest expectation surrounded the inhibition of the KRAS G12C mutation, which occurs in ∼15% of NSCLC, mainly in smokers and is characterized by a poor prognosis. Sotorasib and adagrasib are approved as second-line agents after at least one prior course of chemotherapy and/or immunotherapy. Adagrasib in first-line combination with pembrolizumab immunotherapy proved more beneficial, especially in patients with high expression of PD-L1. In EGFR exon 20 insertion mutation of lung adenocarcinoma, amivantanab was registered for progression after platinum-based chemotherapy. Lung adenocarcinoma carries an EGFR exon 20, HER2 insertion mutation in 2%, for which the first targeted therapy is trastuzumab deruxtecan, in patients already treated with platinum-based chemotherapy. Two orally administered selective c-MET inhibitors, capmatinib and tepotinib, were also approved after chemotherapy in adenocarcinoma carrying MET exon 14 skipping mutations of about 3%. Incorporating reflex testing with next-generation sequencing (NGS) expands personalized therapies by identifying guideline-recommended molecular alterations.

Temporal trends and regional variability in BRAF and KRAS genetic testing in Denmark (2010-2022): Implications for precision medicine.

OBJECTIVE: This study aims to evaluate the developments in the testing of Kirsten Rat Sarcoma viral oncogene homolog (KRAS) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations across different cancer types and regions in Denmark from 2010 to 2022. STUDY DESIGN AND SETTING: Using comprehensive data from the Danish health registries, we linked molecular test results from the Danish Pathology Registry with cancer diagnoses from the Danish National Patient Registry between 2010 and 2022. We assessed the frequency and distribution of KRAS and BRAF mutations across all cancer types, years of testing, and the five Danish regions. RESULTS: The study included records of KRAS testing for 30 671 patients and BRAF testing for 30 860 patients. Most KRAS testing was performed in colorectal (78%) and lung cancer (18%), and BRAF testing in malignant melanoma (13%), colorectal cancer (67%), and lung cancer (12%). Testing rates and documentation mutational subtypes increased over time. Reporting of wildtype results varied between lung and colorectal cancer, with underreporting in lung cancer. Regional variations in testing and reporting were observed. CONCLUSION: Our study highlights substantial progress in KRAS and BRAF testing in Denmark from 2010 to 2022, evidenced by increased and more specific reporting of mutational test results, thereby improving the precision of cancer diagnosis and treatment. However, persistent regional variations and limited testing for cancer types beyond melanoma, colorectal, and lung cancer highlight the necessity for a nationwide assessment of the optimal testing approach.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Genetic Signature of Human Pancreatic Cancer and Personalized Targeting.

Pancreatic cancer is a highly lethal disease with a 5-year survival rate of around 11-12%. Surgery, being the treatment of choice, is only possible in 20% of symptomatic patients. The main reason is that when it becomes symptomatic, IT IS the tumor is usually locally advanced and/or has metastasized to distant organs; thus, early diagnosis is infrequent. The lack of specific early symptoms is an important cause of late diagnosis. Unfortunately, diagnostic tumor markers become positive at a late stage, and there is a lack of early-stage markers. Surgical and non-surgical cases are treated with neoadjuvant and/or adjuvant chemotherapy, and the results are usually poor. However, personalized targeted therapy directed against tumor drivers may improve this situation. Until recently, many pancreatic tumor driver genes/proteins were considered untargetable. Chemical and physical characteristics of mutated KRAS are a formidable challenge to overcome. This situation is slowly changing. For the first time, there are candidate drugs that can target the main driver gene of pancreatic cancer: KRAS. Indeed, KRAS inhibition has been clinically achieved in lung cancer and, at the pre-clinical level, in pancreatic cancer as well. This will probably change the very poor outlook for this disease. This paper reviews the genetic characteristics of sporadic and hereditary predisposition to pancreatic cancer and the possibilities of a personalized treatment according to the genetic signature.

Drugging the undruggable: Advances in targeting KRAS signaling in solid tumors.

Cancer remains the leading cause of global mortality, prompting a paradigm shift in its treatment and outcomes with the advent of targeted therapies. Among the most prevalent mutations in RAS-driven cancers, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations account for approximately 86% of cases worldwide, particularly in lung, pancreatic, and colon cancers, contributing to poor prognosis and reduced overall survival. Despite numerous efforts to understand the biology of KRAS mutants and their pivotal role in cancer development, the lack of well-defined drug-binding pockets has deemed KRAS an "undruggable" therapeutic target, presenting significant challenges for researchers and clinicians alike. Through significant biochemical and technological advances, the last decade has witnessed promising breakthroughs in targeted therapies for KRAS-mutated lung, colon, and pancreatic cancers, marking a critical turning point in the field. In this chapter, we provide an overview of the characteristics of KRAS mutations across various solid tumors, highlighting ongoing cutting-edge research on the immune microenvironment, the development of KRAS-driven mice models, and the recent progress in the exploration of specific KRAS mutant-targeted therapeutic approaches. By comprehensive understanding of the intricacies of KRAS signaling in solid tumors and the latest therapeutic developments, this chapter will shed light on the potential for novel therapeutic strategies to combat KRAS-driven tumors and improve patient outcomes.

KRAS: Biology, Inhibition, and Mechanisms of Inhibitor Resistance.

KRAS is a small GTPase that is among the most commonly mutated oncogenes in cancer. Here, we discuss KRAS biology, therapeutic avenues to target it, and mechanisms of resistance that tumors employ in response to KRAS inhibition. Several strategies are under investigation for inhibiting oncogenic KRAS, including small molecule compounds targeting specific KRAS mutations, pan-KRAS inhibitors, PROTACs, siRNAs, PNAs, and mutant KRAS-specific immunostimulatory strategies. A central challenge to therapeutic effectiveness is the frequent development of resistance to these treatments. Direct resistance mechanisms can involve KRAS mutations that reduce drug efficacy or copy number alterations that increase the expression of mutant KRAS. Indirect resistance mechanisms arise from mutations that can rescue mutant KRAS-dependent cells either by reactivating the same signaling or via alternative pathways. Further, non-mutational forms of resistance can take the form of epigenetic marks, transcriptional reprogramming, or alterations within the tumor microenvironment. As the possible strategies to inhibit KRAS expand, understanding the nuances of resistance mechanisms is paramount to the development of both enhanced therapeutics and innovative drug combinations.