Investigating A Novel Mechanism of Farnesyl Transferase Inhibitors in HRAS Cancers

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Rare cancers, combined, account for over half of all cancer diagnoses per year. Cancer-causing mutations in the three major RAS proteins, H-, N-, and KRAS4B, are common – with mutations in HRAS being the rarest. Broadly, RAS proteins act as molecular switches for proliferation by cycling between GDP- (off) and GTP-bound (on) states. Oncogenic mutations in RAS favor a higher GTP/GDP ratio and often result in the inhibition of one of RAS’ key regulators, NF1, by forming a stoichiometric sink that leads to constitutive signal.

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One of the earliest attempts for a targeted therapy against mutant RAS was an inhibitor against farnesyl transferase. Farnesyl transferase is an enzyme that adds a post-translational modification on nascent-RAS proteins that is responsible for membrane localization – without which, they are stuck in the cytoplasm and lack efficient signaling. By inhibiting farnesyl transferase, farnesyl transferase inhibitors (FTIs) reduce plasma-membrane localization of RAS and therefore inhibit oncogenic signaling. However, FTIs failed clinical trials due to lack of efficacy. Post-mortem insights revealed that much of the pre-clinical work in FTIs was done in mutant-HRAS cancers, while clinical trials were largely performed in mutant-KRAS cancers. This mattered, because KRAS4B was discovered to be processable by another enzyme, geranylgeranyl transferase, that compensates for loss of farnesyl transferase. Further, KRAS4B has a higher affinity for farnesyl transferase than H/NRAS and can effectively out-compete the FTI – making a competitive inhibitor dually obsolete. H/NRAS-membrane localization, however, is effectively inhibited by FTIs.

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Pre-clinical studies still suggest that FTIs can be effective – but perhaps only against mutant H/NRAS cancers. Because HRAS is the most susceptible to FTI treatment, clinical trials for FTIs in mutant-HRAS contexts were re-initiated. Due to the rarity of mutant-HRAS cancers, patients over-expressing HRAS – which is more common than harboring an activating mutation in HRAS – were included. Yet again, resistance to FTIs was observed. Our hypothesis is that the anti-proliferative effect observed in pre-clinical studies is a result of a more subtle mechanism of action: FTIs sequester mutant HRAS in the cytoplasm, where cytosolic, downstream effectors – such as RAF – recognize and bind it. The cytosolic, mutant-HRAS forms a stoichiometric sink on downstream effectors and becomes a dominant-negative regulator of collective-RAS signaling. Therefore, we hypothesize that patients harboring activating mutations in HRAS are likely to respond to FTI treatment, but patients over-expressing wild-type HRAS will not. This project focuses on redefining FTIs’ mechanism of action to benefit patients with rare cancers.

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Understanding the Resistance Mechanism to KRAS G12C Inhibitors in NSCLC and CRC

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For years, mutant RAS was considered undruggable – due to it lacking an obvious binding pocket and because it utilizes GTP instead of ATP (which excludes the possibility of ATP-competitive inhibitors). Eventually, covalent inhibitors of KRAS G12C were developed by targeting the sulfur within KRAS G12C’s cysteine residue. The inhibitors target, and effectively lock, KRAS G12C in its GDP-bound state, thus reducing signal from oncogenic RAS. Initially, G12C inhibitors showed great success in NSCLC but demonstrated less efficacy in CRC. Further, even in NSCLC contexts with homozygous KRAS G12C/G12C mutations, resistance has been observed. One initial hypothesis for this resistance is that upstream receptor tyrosine kinases and/or guanidine exchange factors are increasing GTP loading on RAS, thus conferring resistance to the covalent inhibitor. We hypothesize, however, that the major mechanism of resistance to covalent KRAS G12C inhibitors results from RAS stoichiometry (specifically, a low mutant/WT RAS ratio).

 

Historically, despite over 50 common, different RAS mutations, all activating-RAS mutations were considered synonymous. Growing evidence suggests that there are nuances between each of these mutations, and these differences provide exploitable vulnerabilities. KRAS G12C mutations remain under-characterized as to their biochemical nuances and how these nuances can be exploited. Computationally, KRAS G12C is predicted to have moderate activity against RAS GAPs, to cycle between GTP and GDP, and to have minimal-to-modest interaction with guanidine exchange factors. These middling effects best explain how resistance is developed in NSCLC and CRC when using stoichiometry as a predictor. To better predict and address intrinsic and acquired resistance, KRAS G12C proteins need to be better characterized. This study aims to better characterize and measure KRAS G12C’s contribution to collective-RAS signaling.

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Elucidating the Allele-Specific Differences of Oncogenic RAS Mutations

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RAS mutations most commonly occur at hotspot codons 12, 13, and 61 which are regions critical for GTP hydrolysis and proper regulation of GTPase activity. However, RAS mutations are not created equal; therefore, exhibiting distinct biochemical behaviors within their signaling pathways. 

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This study focuses on understanding how individual RAS mutants behave within the MAPK signaling pathway, with a primary focus on their crosstalk with wildtype RAS counterparts and how these interactions influence proliferative strength driving oncogenesis. Prior work from our lab has demonstrated that RAS mutants differ in their interactions with key regulators which includes GEFs, GAPs, and RTKs. These biochemical differences can confer sensitivity or resistance to therapeutic modalities such as small molecule inhibitors.

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A key example of this is our work on KRAS G12R, where we elucidated a mechanism by which this mutant is unable to regulate wildtype RAS, revealing a therapeutic vulnerability that can be exploited through EGFR or MEK inhibitor treatment to reduce proliferative signaling and achieve tumor regression in vitro and in vivo. This finding exemplifies how understanding the individual mechanisms of each RAS mutant can directly inform precision medicine approaches.

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With the continued advancement of drugs that selectively target RAS in mutant-specific or activated forms, we are now positioned to conduct more extensive network analyses of these mutants within their specific cellular contexts. Our goal is to establish detailed mechanistic frameworks for additional RAS mutants, building upon the foundation of our previously published work to ultimately guide more effective, mutation-specific therapeutic strategies for patients.

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Magnetic 3D printing

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Standard 2D cell culture, while widely used, fails to recapitulate the complex architecture and signaling dynamics of living tumors. Magnetic 3D (m3D) cell culture is a high-throughput, rapid technology that generates physiologically relevant tumor models by mimicking the in vivo environment making it a powerful translational tool for assessing drug effects faster and more meaningfully than traditional 2D methods.

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Our lab utilizes NanoShuttle-PL (Greiner Bio), a biocompatible nanoparticle assembly consisting of gold, iron oxide, and poly-L-lysine (PLL), to magnetize cells via nanoparticle uptake. These metals do not induce toxicity, inflammation, or oxidative stress, and are naturally released into the extracellular matrix after approximately 8 days. Once magnetized, cells undergo magnetic levitation to promote cell-cell interactions and extracellular matrix formation, then are seeded into cell-repellent plates above magnetic drives, where they self-assemble into precise 3D structures within 2 hours.​ Our lab also employs ring drives to generate donut-shaped spheroids and dot drives to generate smaller donut-hole structures, requiring a fraction of the cell number. These structures are monitored over time via microscopy and LiCor imaging, where closure or expansion of the ring serves as a direct readout of drug response. We also utilize 3D cell viability to obtain quantitative measurements of effects on proliferation.

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We apply this platform across multiple cancer types, screening inhibitors tailored to each tumor's mutational profile. Notably, our lab has successfully printed dissociated patient tumor samples for drug screening, bridging the gap between bench findings and clinical relevance. We are also actively optimizing rapid organoid formation using this technology. Ultimately, m3D culture serves as an orthogonal approach to validate our 2D findings while enabling faster, more translationally relevant drug screening across a broader range of cancer types and RAS mutants.

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