Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

HRAS-driven cancer cells are vulnerable to TRPML1 inhibition.

By serving as intermediaries between cellular metabolism and the bioenergetic demands of proliferation, endolysosomes allow cancer cells to thrive under normally detrimental conditions. Here, we show that an endolysosomal TRP channel, TRPML1, is necessary for the proliferation of cancer cells that bear activating mutations in HRAS Expression of MCOLN1, which encodes TRPML1, is significantly elevated in HRAS-positive tumors and inversely correlated with patient prognosis. Concordantly, MCOLN1 knockdown or TRPML1 inhibition selectively reduces the proliferation of cancer cells that express oncogenic, but not wild-type, HRAS Mechanistically, TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport. TRPML1 inhibition disrupts the distribution and levels of cholesterol and thereby attenuates HRAS nanoclustering and plasma membrane abundance, ERK phosphorylation, and cell proliferation. These findings reveal a selective vulnerability of HRAS-driven cancers to TRPML1 inhibition, which may be leveraged as an actionable therapeutic strategy.

The impact of coronavirus disease 2019 pandemic on dental school assessments - Current status and future perspectives.

PURPOSE: To evaluate course directors' feedback on the assessment methods used during the coronavirus disease 2019 (COVID-19) pandemic and identify effective approaches for future assessments in dental education. METHODS: Course directors at the US dental schools were surveyed for changes in assessments implemented during the early stages of the pandemic (March-July 2020) using the Qualtrics platform. The survey questions addressed assessment methods utilized in didactic, preclinical, and clinical arenas pre-COVID-19 (before March 2020) and during the early phase of the pandemic (between March and July 2020) and identified any sustained changes in assessments post-COVID-19. Of the 295 responses for the type of courses directed, 48%, 22%, and 30% responses were for didactic, pre-clinical, and clinical assessments, respectively. Chi-square tests and 95% confidence intervals were used to assess quantitative differences. RESULTS: Computer-based un-proctored and remote- proctored assessments increased whereas paper-based in-person proctored assessments decreased during an early pandemic. For pre-clinical and clinical courses, objective-structured clinical exams and case-based assessments increased whereas, for didactic courses, the number of presentations, short-answer, and multiple-choice questions-based assessments increased. Specimen-based assessments and patient-based encounters decreased significantly in didactic and clinical courses, respectively. Manikin-based exams increased in clinical but not in pre-clinical courses. Survey respondents disagreed that alternative assessments helped students learn better, resulted in better course evaluations, or were an equivalent replacement for pre-COVID-19 assessments. Interestingly, 49% of respondents indicated a likelihood of continuing alternative assessments whereas 36% were unlikely and 15% were neutral. CONCLUSIONS: A combination of effective pre-pandemic and innovative alternative assessments developed during the pandemic may be the new normal in the dental education curriculum.

Evaluating dental students' perspectives on the concurrent teaching of didactic and case-based courses.

PURPOSE: To evaluate student perception of integrating biomedical and clinical sciences based on survey of dental students on the concurrent teaching of a didactic systems-based course and a case-based course. METHODS: First-year to fourth-year students (DS1-DS4) students were surveyed for their experiences in concurrent teaching. Student response rate for the survey was 55% (229/420). Pearson's Chi-squared tests and Kruskal-Wallis rank sum tests were used to assess statistical significance (p < 0.05). RESULTS: Of the students surveyed, 83% strongly agreed or agreed that concurrent teaching of the didactic and case-based courses helped them better understand the biomedical science background and the clinical ramifications (p < 0.001). On average, 75% percent strongly agreed or agreed that concurrent teaching kept them engaged, motivated, think critically, apply the course content and prepare for clinical practice (p < 0.001). Of the students surveyed, 69% support expanding concurrent teaching to all four years (p < 0.001). Mean responses from DS1 and DS4 students differed for questions relating to understanding of biomedical sciences, critical thinking and application to clinic (p < 0.01). Qualitative data showed that students enjoyed the reinforcement of concepts and application to clinical scenarios. CONCLUSIONS: Concurrent teaching of didactic and case-based learning courses, thus showing clinical relevance of biomedical sciences in the first year of dental curriculum, is perceived by students as an effective method of educating dental students. Such integrative learning process with horizontal and vertical integration and concurrent curriculum is even more relevant with the implementation of the integrated national board dental examination.

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils.

The formation of adenosine dampens inflammation by inhibiting most cells of the immune system. Among its actions on neutrophils, adenosine suppresses superoxide generation and regulates chemotactic activity. To date, most evidence implicates the G(s) protein-coupled A(2A) adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A(2B)AR is now known to be expressed in neutrophils and that it is a G(s) protein-coupled receptor, we examined in this study whether it signals to suppress neutrophil activities by using 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY 60-6583), a new agonist for the human A(2B)AR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A(2B)AR. We found that treating mouse neutrophils with low concentrations (10(-9) and 10(-8) M) of BAY 60-6583 inhibited formylated-methionine-leucine-phenylalanine (fMLP)-stimulated superoxide production by either naive neutrophils, tumor necrosis factor-α-primed neutrophils, or neutrophils isolated from mice treated systemically with lipopolysaccharide. This inhibitory action of BAY 60-6583 was confirmed to involve the A(2B)AR in experiments using neutrophils obtained from A(2B)AR gene knockout mice. It is noteworthy that BAY 60-6583 increased fMLP-stimulated superoxide production at higher concentrations (>1 µM), which was attributed to an AR-independent effect. In a standard Boyden chamber migration assay, BAY 60-6583 alone did not stimulate neutrophil chemotaxis or influence chemotaxis in response to fMLP. These results indicate that the A(2B)AR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A(2A)AR in regulating the proinflammatory actions of neutrophils.

Scaffold repurposing of fendiline: Identification of potent KRAS plasma membrane localization inhibitors.

KRAS plays an essential role in regulating cell proliferation, differentiation, migration and survival. Mutated KRAS is a major driver of malignant transformation in multiple human cancers. We showed previously that fendiline (6) is an effective inhibitor of KRAS plasma membrane (PM) localization and function. In this study, we designed, synthesized and evaluated a series of new fendiline analogs to optimize its drug properties. Systemic structure-activity relationship studies by scaffold repurposing led to the discovery of several more active KRAS PM localization inhibitors such as compounds 12f (NY0244), 12h (NY0331) and 22 (NY0335) which exhibit nanomolar potencies. These compounds inhibited oncogenic KRAS-driven cancer cell proliferation at single-digit micromolar concentrations in vitro. In vivo studies in a xenograft model of pancreatic cancer revealed that 12h and 22 suppressed oncogenic KRAS-expressing MiaPaCa-2 tumor growth at a low dose range of 1-5 mg/kg with no vasodilatory effects, indicating their potential as chemical probes and anticancer therapeutics.

A(3) adenosine receptor activation during reperfusion reduces infarct size through actions on bone marrow-derived cells.

The goal of this study was to examine whether the A(3) adenosine receptor (A(3)AR) agonist Cl-IB-MECA protects against myocardial ischemia/reperfusion injury when administered at the time of reperfusion in an in vivo mouse model of infarction induced by 30min of coronary occlusion and 24h of reperfusion. Treating B6 wild-type with Cl-IB-MECA during the reperfusion phase (100microg/kg i.v. bolus+0.3microg/kg/min subcutaneously via implantation of Alzet mini-osmotic pumps) reduced myocardial infarct size approximately 37% from 50.1+/-2.5% in vehicle-treated mice to 31.6+/-2.8% in Cl-IB-MECA-treated mice, and significantly reduced the number of leukocytes that infiltrated into the ischemic-reperfused myocardium. Cl-IB-MECA did not reduce infarct size or limit leukocyte accumulation in studies using B6 congenic A(3)AR gene "knock-out" mice or in chimeric mice lacking the expression of A(3)ARs in bone marrow (BM)-derived cells. Subsequent mechanistic studies demonstrated that Cl-IB-MECA inhibited migration of mouse neutrophils isolated from BM towards the chemotactic substance c5a in trans-well migration assays, and inhibited leukocyte migration into the peritoneal cavity in a mouse model of thioglycollate-induced peritonitis. We conclude that treating with the A(3)AR agonist Cl-IB-MECA at the time of reperfusion provides effective protection from ischemia/reperfusion injury in the heart through activation of the A(3)AR expressed in BM-derived cells, potentially by suppressing the robust inflammatory reaction that occurs during reperfusion and neutrophil-mediated tissue injury.

Nucleophosmin and nucleolin regulate K-Ras plasma membrane interactions and MAPK signal transduction.

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.

Assessment of Critical Thinking in a First-Year Dental Curriculum.

We studied how students in a Doctor of Dental Surgery (DDS) program perceived the importance of critical thinking and the extent to which critical thinking was perceived to be included in each of 25 courses of the first-year curriculum at The University of Texas School of Dentistry at Houston (UTSD). Sixty-nine of the 102 second-year students who were invited participated in an online survey. The survey had three parts, with all statements of each part evaluated on a five-point Likert scale ranging from strongly agree to strongly disagree. The first two parts assessed the importance of critical thinking in dental education and the criteria by which critical thinking in didactic curriculum can be measured. In the third part of the survey, students evaluated how well each course of the first-year curriculum achieved critical thinking. More than 90% of the respondents strongly agreed/agreed that critical thinking is essential to making clinical decisions. Students strongly agreed/agreed that 19 of 25 of the courses incorporated critical thinking. However, when students were asked to rank the top five of the 25 courses, only two courses (Human Biology, Neuroscience) emerged above all others in their weighted ranks, with another seven courses standing out, leaving 16 courses with low weighted rankings for their inclusion of critical thinking. In summary, students agreed on the importance of critical thinking in dental education, and on the criteria by which the incorporation of critical thinking should be measured in didactic and pre-clinical courses.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Methods and timing of curricular integration in U.S. dental education in preparation for the Integrated National Board Dental Examination.

PURPOSE: To characterize methods, timing, faculty training, and barriers pertaining to curricular integration in preparation for the Integrated National Board Dental Examination (INBDE) at U.S. dental schools METHODS: Academic deans at 67 U.S. dental schools were emailed an online survey consisting of questions on the time frame and methods of curricular integration, faculty training, and barriers to curricular integration RESULTS: Approximately 86% of schools have implemented (∼39%) or will implement (∼61%) changes in curriculum in preparation for the INBDE. Curricular integration was completed in 3-4 years in most schools, with those schools with larger class sizes taking longer. While 56% of respondents reported avoiding a complete curricular overhaul, 79% reported course sequencing changes, adding, subtracting, or combining courses, and making some changes in existing courses. Curriculum committees had the most input in curricular changes, while individual instructors had the least influence. Sixty seven percent of respondents reported faculty being trained to teach in an integrated curriculum. Problem/case-based learning and clinical case presentations are being used more for incorporation of biomedical sciences in clinical curriculum; use of clinical examples was the most used mechanism to integrate clinical information in biomedical sciences curriculum. Eighty two percent of respondents indicated that lack of faculty time to prepare courses as the primary barrier for implementing an integrated curriculum. CONCLUSIONS: Our results show that most U.S. dental schools are implementing integrated teaching models to prepare their students for the INBDE. However, lack of faculty time and training need to be addressed for more successful curricular integration.

Integration of Basic and Clinical Sciences: Student Perceptions.

The integrated curriculum is becoming a popular concept among dental schools. The purpose of this study was to query dental students at the University of Texas Health Science Center at Houston - School of Dentistry (UTSD) to elucidate their level of interest in the integrated curriculum, perception of how much integration is currently occurring, and identify challenges to integration. To address this question, dental students at UTSD were invited to participate in a survey. Participants reported their perspectives on integration of sciences. All survey participants agreed that it is beneficial to integrate clinical and basic sciences and that basic science educators were incorporating clinical relevance in their regular teaching. The third and fourth year classes, classes that had been exposed to general as well as all specialty dentistry clinics, agreed that basic sciences are being incorporated into most clinical teaching. Top two barriers to integration identified by the students were lack of crossover knowledge of faculty, and insufficient time to explore connections between basic sciences and clinical sciences because of the volume of information that needs to be covered. In conclusion, student perception at UTSD is that overall basic and clinical sciences are being integrated throughout the curriculum.

Characterization of the A2B adenosine receptor from mouse, rabbit, and dog.

We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [(3)H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-(3)H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([(3)H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.

Discovery of High-Affinity Noncovalent Allosteric KRAS Inhibitors That Disrupt Effector Binding.

Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.

Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function.

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

Activation of the A(3) adenosine receptor suppresses superoxide production and chemotaxis of mouse bone marrow neutrophils.

Adenosine is formed in injured/ischemic tissues, where it suppresses the actions of essentially all cells of the immune system. Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the G(s) protein-coupled A(2A) adenosine receptor (AR). Here, we report that the A(3)AR is highly expressed in murine neutrophils isolated from bone marrow. Selective activation of the A(3)AR with (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903) potently inhibited mouse bone marrow neutrophil superoxide generation and chemotaxis induced by various activating agents. The selectivity of CP-532,903 was confirmed in assays using neutrophils obtained from A(2A)AR and A(3)AR gene "knockout" mice. In a model of thioglycollate-induced inflammation, treating mice with CP-532,903 inhibited recruitment of leukocytes into the peritoneum by specifically activating the A(3)AR. Collectively, our findings support the theory that the A(3)AR contributes to the anti-inflammatory actions of adenosine on neutrophils and provide a potential mechanistic explanation for the efficacy of A(3)AR agonists in animal models of inflammation (i.e., inhibition of neutrophil-mediated tissue injury).

Integration of Basic and Clinical Sciences: Faculty Perspectives at a U.S. Dental School.

Although dental education has traditionally been organized into basic sciences education (first and second years) and clinical education (third and fourth years), there has been growing interest in ways to better integrate the two to more effectively educate students and prepare them for practice. Since 2012, The University of Texas School of Dentistry at Houston (UTSD) has made it a priority to improve integration of basic and clinical sciences, with a focus to this point on integrating the basic sciences. The aim of this study was to determine the perspectives of basic and clinical science faculty members regarding basic and clinical sciences integration and the degree of integration currently occurring. In October 2016, all 227 faculty members (15 basic scientists and 212 clinicians) were invited to participate in an online survey. Of the 212 clinicians, 84 completed the clinician educator survey (response rate 40%). All 15 basic scientists completed the basic science educator survey (response rate 100%). The majority of basic and clinical respondents affirmed the value of integration (93.3%, 97.6%, respectively) and reported regular integration in their teaching (80%, 86.9%). There were no significant differences between basic scientists and clinicians on perceived importance (p=0.457) and comfort with integration (p=0.240), but the basic scientists were more likely to integrate (p=0.039) and collaborate (p=0.021) than the clinicians. There were no significant differences between generalist and specialist clinicians on importance (p=0.474) and degree (p=0.972) of integration in teaching and intent to collaborate (p=0.864), but the specialists reported feeling more comfortable presenting basic science information (p=0.033). Protected faculty time for collaborative efforts and a repository of integrated basic science and clinical examples for use in teaching and faculty development were recommended to improve integration. Although questions might be raised about the respondents' definition of "integration," this study provides a baseline assessment of perceptions at a dental school that is placing a priority on integration.

Sphingomyelin Metabolism Is a Regulator of K-Ras Function.

K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in Caenorhabditis elegans, as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against C. elegans genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.